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Intra- and intermolecular interactions in proteins Studies of marginally hydrophobic transmembrane α-helices and protein-protein interactions. Linnea E. Hedin

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Intra- and intermolecular interactions in proteins Studies of marginally hydrophobic transmembrane α-helices

and protein-protein interactions.

Linnea E. Hedin

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© Linnea E. Hedin, Stockholm 2010

ISBN 978-91-7447-111-3

Printed in Sweden by US-AB, Stockholm 2010

Distributor: Department of Biochemistry and Biophysics, Stockholm University

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To my parents

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Abstract

Most of the processes in a living cell are carried out by proteins.

Depending on the needs of the cell, different proteins will interact

and form the molecular machines demanded for the moment. A

subset of proteins called integral membrane proteins are responsible

for the interchange of matter and information across the biological

membrane, the lipid bilayer enveloping and defining the cell. Most

of these proteins are co-translationally integrated into the membrane

by the Sec translocation machinery.

This thesis addresses two questions that have emerged during the

last decade. The first concerns membrane proteins: a number of α-

helices have been observed to span the membrane in the obtained

three-dimensional structures even though these helices are predicted

not to be hydrophobic enough to be recognized by the translocon for

integration. We show for a number of these marginally hydrophobic

protein segments that they indeed do not insert well outside of their

native context, but that their local sequence context can improve the

level of integration mediated by the translocon. We also find that

many of these helices are overlapped by more hydrophobic

segments. We propose, supported by experimental results, that the

latter are initially integrated into the membrane, followed by post-

translational structural rearrangements. Finally, we investigate

whether the integration of the marginally hydrophobic TMHs of the

lactose permease of Escherichia coli is facilitated by the formation

of hairpin structures. However our combined efforts of

computational simulations and experimental investigations find no

evidence for this.

The second question addressed in this thesis is that of the

interpretation of the large datasets on which proteins that interact

with each other in a cell. We have analyzed the results from several

large-scale investigations concerning protein interactions in yeast

and draw conclusions regarding the biases, strengths and weaknesses

of these datasets and the methods used to obtain them.

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Populärvetenskaplig sammanfattning

Av alla de molekyler som finns i och bygger upp en cell är det

proteinerna som är de verkliga arbetshästarna och som står för de

flesta processer som äger rum i cellen. Cellens behov varierar

kontinuerligt, och olika proteiner interagerar med varandra för att

bilda de molekylära verktyg som behövs för stunden.

En undergrupp av proteiner som kallas membranproteiner

ansvarar för utbyte av materia (näring, etc) och information över det

biologiska membranet, det dubbla lager av lipider som omsluter och

definierar cellen. Många sjukdomar är sammankopplade med

membranproteiner som inte fungerar som de ska och studier av

membranproteiner är av högsta vikt för att kunna förstå och bota

dessa sjukdomar.

De flesta membranproteiner sätts in i lipidbilagret samtidigt som

de syntetiseras, via ett maskineri som kallas Sec-translokonet.

Proteinernas byggstenar, aminosyrorna, har olika kemiska

egenskaper. För att kännas igen av translokonet måste ett

proteinsegment bestå av aminosyror som är hydrofoba, som löser sig

bättre i fett än i vatten.

Den här avhandlingen tar upp två frågor som uppstått det senaste

decenniet: Den första frågan rör de proteinsegment som är trädda

genom membranet. Enligt vad som är känt idag är vissa av dessa

segment inte tillräckligt hydrofoba för att kännas igen av

translokonet och stoppas in i membranet. Trots detta syns det tydligt

i tredimensionella bilder av membranproteiner att dessa marginellt

hydrofoba segment faktiskt är inbäddade i membranet. Vi har

undersökt hur detta kan förklaras.

Vi visar för ett antal av dessa segment att de i sig själva inte är

tillräckliga för att effektivt sättas in i membranet, men att egenskaper

hos de angränsande proteinsegmenten kan vara tillräckliga för att få

dem att sättas in. Vi finner också att många av dessa mindre

hydrofoba segment överlappas av mer hydrofoba dito. Vi presenterar

resultat som föreslår att translokonet initialt känner igen de mer

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hydrofoba, överlappande segmenten och att den slutliga strukturen

fås genom att proteinet skiftar sin position i membranet efter att

proteinsyntesen avslutats.

Slutligen undersöker vi proteinet laktospermeas och huruvida

dess många marginellt hydrofoba segment sätts in i membranet på

grund av täta interaktioner med det närmaste föregående eller

efterföljande membraninbäddade segmentet. Vi använder både

datorsimuleringar och experimentella undersökningar, men hittar

inget som talar för det ovanstående.

Den andra frågan rör tolkningen av den stora mängden

information om vilka proteiner som interagerar med varandra. Vi har

analyserat resultaten från flera storskaliga undersökningar av

interaktioner i jästceller och drar slutsatser angående styrkorna och

svagheterna i dessa dataset samt de metoder som använts för att ta

fram dem.

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List of papers

This thesis is based on the following publications:

Paper I

Paper II

Paper III

Paper IV

Hedin LE*, Öjemalm K*, Bernsel A, Hennerdal A,

Illergård K, Enquist K, Kauko A, Cristobal S, von Heijne

G, Lerch-Bader M, Nilsson I, Elofsson A. “Membrane

insertion of marginally hydrophobic transmembrane

helices depends on sequence context.” (2010) Journal of

Molecular Biology 396(1):221-9

Kauko A*, Hedin LE*, Thebaud E, Cristobal S, Elofsson

A, von Heijne G. “Repositioning of transmembrane

alpha-helices during membrane protein folding.” (2010)

Journal of Molecular Biology 397(1):190-201

Vereshchaga Y*, Hedin L*, Cristobal S, Elofsson A,

Lindahl E. “Insertion Properties of Marginally

Hydrophobic Helices in the LacY Lactose Permease

Transporter” (Manuscript.)

Björklund AK, Light S, Hedin L, Elofsson A.

“Quantitative assessment of the structural bias in protein-

protein interaction assays.” (2008) Proteomics

8(22):4657-67

* Equal contribution

Reprints were made with permissions from the publishers.

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Contents

Abbreviations

Chapter 1 Introduction ....................................................................... 13

Chapter 2 Biological membranes ....................................................... 15

2.1 Biological membranes define the cell ........................................ 15

2.2 Biological membranes form due to the hydrophobic effect ....... 16

2.3 The hydrophobic effect is caused by water ................................ 16

2.4 Basic structure of phospholipid membranes .............................. 17

2.5 Biological membranes consist of more than lipids .................... 18

Chapter 3 Proteins in general ............................................................ 19

3.1 Protein structure and folding in general ..................................... 19

3.2 More on the quartenary structure of proteins, protein-protein

interactions ................................................................................ 21

3.3 Evolutionary evolved functional substructures, protein domains

................................................................................................... 22

Chapter 4 Membrane proteins, what do they look like? .................. 23

4.1 Structure and topology of α-helical membrane proteins ............ 24

4.1.1 The primary structure of a transmembrane helix is adapted to

its surroundings .................................................................... 24

4.1.2 Secondary structure in transmembrane segments can be

irregular ................................................................................ 25

4.1.3 Other structural elements of helical membrane proteins ...... 26

4.2 Forces and motifs implied in the folding of helical membrane

proteins ...................................................................................... 26

4.2.1 Intramolecular forces stabilizing the native fold .................. 27

4.2.2 Lipid-protein interactions affecting the folded protein 28

Chapter 5 Biogenesis and bilayer integration of α-helical membrane

proteins .............................................................................. 31

5.1 The Sec translocon channel ....................................................... 32

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5.2 The versatile translocon ............................................................. 33

5.3 Other systems for insertion of membrane proteins .................... 34

Chapter 6 How is topology decided during biogenesis and folding?

........................................................................................... 37

6.1 Charged residues in loops, the positive inside rule .................... 38

6.2 Hydrophobicity as recognized by the translocon ....................... 39

6.3 Marginally hydrophobic transmembrane helices do exist.......... 39

6.4 Length of the hydrophobic segment .......................................... 40

6.5 Reentrant loops .......................................................................... 41

6.6 Other factors .............................................................................. 41

Chapter 7 Structure analysis ............................................................. 43

7.1 Obtaining three-dimensional structures ..................................... 43

7.2 In silico investigations ............................................................... 44

7.3 Determining membrane protein topology experimentally ......... 45

7.3.1 N-linked glycosylation as topology reporter and molecular

ruler ...................................................................................... 46

7.4 Determining the propensity to integrate into the membrane ...... 46

7.4.1 Model proteins, LepI and LepII ........................................... 47

7.4.2 In vitro protein expression ................................................... 48

7.5 Determining quartenary protein structure, the composition of

protein complexes ...................................................................... 48

Chapter 8 Summary of the papers .................................................... 51

8.1 Paper I. ....................................................................................... 51

8.2 Paper II. ..................................................................................... 52

8.2 Paper III. .................................................................................... 52

8.4 Paper IV. .................................................................................... 53

8.5 Final thoughts ............................................................................ 55

Acknowledgements ............................................................................. 57

Bibliography ........................................................................................ 59

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Abbreviations

DOPC

ER

LC

MALDI-TOF

MP

MS/MS

mTMH

RM

SDS-PAGE

TM

TMH

Å

1,2-dioleoyl-sn-glycero-3-phosphocholine

Endoplasmic Reticulum

Liquid Chromatography

Matrix Assisted Laser Desorption/Ionization – Time

of Flight

Membrane Protein

Tandem Mass Spectrometry Marginally hydrophobic Transmembrane Helix

Rough Microsomes

Sodium dodecyl sulphate polyacrylamide gel

electrophoresis

Transmembrane

Transmembrane Helix

Ångström, 1 Å = 0.1 nm

Amino acids, one and three letter codes:

Any amino acid - Xaa - X Leucine - Leu - L

Alanine - Ala - A Lysine - Lys - K

Arginine - Arg - R Methionine - Met - M

Asparagine - Asn - N Phenylalanine - Phe - F

Aspartic Acid - Asp - D Proline - Pro - P

Cysteine - Cys - C Serine - Ser - S

Glutamine - Gln - Q Threonine - Thr - T

Glutamic acid - Glu - E Tryptophan - Trp - W

Glycine - Gly - G Tyrosine - Tyr - Y

Histidine - His - H Valine - Val - V

Isoleucine - Iso - I

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13

Chapter 1

Introduction

The basic building blocks of life are water, nucleotides (DNA, RNA), lipids

and amino acids. Throw in some ions and sugars and you are pretty much set.

DNA carries the information, RNA helps putting the information to use, lipids

make up the biological membranes that separate a cell from the rest of the

world and the cellular organelles from the rest of the cell. Amino acids are put

together into proteins according to the information encoded in the DNA.

These proteins can in turn form larger complexes, varying in composition

depending on the function. Proteins are what make a cell work; they are the

workhorses and small factories of the cell. They give structure and mobility

(cytoskeleton) and take care of molecular synthesis and degradation. They are

involved in energy metabolism and allow exchange of information and

material both within the cell and with the outside (receptors, transporters,

channels), etc. Especially the latter tasks involve a certain category of

proteins, integral membrane proteins. It is easy to imagine that malfunctioning

membrane proteins can result in severe problems for a cell and, in a

multicellular being, the whole organism. In fact, more than half of the targets

in the pharmaceutical industry are membrane proteins1; 2

. To understand how a

chain of amino acids folds properly and gets integrated into the lipid bilayer is

thus an important problem to solve, and not only out of pure curiosity.

This thesis addresses the question of how less hydrophobic protein

segments get incorporated into the membrane. It also deals with large-scale

investigations of which proteins form complexes together and the importance

of knowing about method biases when interpreting results from these

investigations.

I will start by presenting a general picture of a biological membranes and the

proteins within them, what they look like and why. I will then give a short

overview of the major machinery needed to insert proteins into biological

membranes and the factors affecting the insertion process. I will also briefly

review other machineries for insertion and translocation. Thereafter I discuss

ways to study protein structure, including how to study quaternary structure by

deciphering the composition of protein complexes. In the end the articles and

the work included in this thesis are summed up and finally I share some final

thoughts on the subject.

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15

Chapter 2

Biological membranes

This chapter gives a brief introduction to biological membranes, the tiny

barriers that distinguish cells from the rest of the world.

2.1 Biological membranes define the cell

Cells are the basic unit of life, defined and demarcated by their cell

membranes. Most unicellular organisms manage all the processes needed to

live and propagate within one cell, while the vast majority of multicellular

organisms are composed by differentiated cells specializing in different

functions. From an evolutionary point of view, all living organisms can be

divided into three major domains, or kingdoms: Bacteria, Archaea and

Eukarya. This division is often useful when discussing different molecular

aspects of life. Bacteria and archaeans are generally simpler than eukaryotes.

For instance, bacteria and archaea are most often unicellular while

eukaryotes usually are multicellular. There are however exceptions like

facultative multicellular prokaryotes3; 4

, colonial cyanobacteria5 and the

eukaryotic, unicellular yeasts and protists. Not only are most eukaryotes

comprised by cells specialized in different ways, but all eukaryotic cells have

organelles, internal compartments which are enclosed by membranes and

specialize in different cellular functions, see figure 1. Organelles in Archaea

and Bacteria are much rarer, but some examples do exist6; 7

.

Figure 1. A schematic

picture of an animal cell.

ER, Endoplasmic reti-

culum. Adapted from

http://en.wikipedia.org/wik

i/File:Biological_cell.svg,

accession date July 2010.

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16 Chapter 2 • Biological membranes

The exact processes taking place in each organelle can vary between

different cell types, but these are commonly the major tasks: Peroxisomes

and lysosomes specialize in synthesis and breakdown of macromolecules,

the nucleus in replication and transcription of DNA, the smooth endoplasmic

reticulum in a wide range of metabolic reactions, the rough endoplasmic

reticulum in protein synthesis, and the Golgi in protein modifications.

Mitochondria have two membranes, one inner and one outer, and specialize

in energy conversion. Chloroplasts, the photosynthetic plant organelles (not

shown here) also have double membranes and even have an organelle of

their own, the thylakoid. The thylakoids is responsible for converting

electromagnetic energy (light) into chemical energy.

2.2 Biological membranes form due to the hydrophobic

effect

Membranes exist, and thus cells and thus you yourself, due to the same

principle that makes oil and water mix poorly. Fatty, or hydrophobic, “water-

fearing” surfaces in an aqueous environment will end up interacting with

each other. This is called the hydrophobic effect, and it drives the formation

of biological membranes and the folding of a chain of amino acids into a

functional protein8. Phospholipids, the basic building blocks of a biological

membrane, have one hydrophobic and one hydrophilic, “water-loving” end

and will therefore form bilayers.

2.3 The hydrophobic effect is caused by water

The hydrophobic effect is in truth more of a hydrophilic effect. Due to the

high electronegativity of oxygen and the small size of the hydrogen atom,

water molecules are electric dipoles, with a partial negative charge on the

oxygen atom and a partial positive charge on each hydrogen atom. Water

molecules will therefore engage in hydrogen bonds, strong electrostatic

interactions (see paragraph 4.2), with each other and other polar molecules.

When a non-polar, hydrophobic entity is placed in water, it either falls out of

solution as it cannot interact electrostatically with the solvent, or the water

molecules form a cage-like structure around it, keeping it solubilized, see

figure 2. This structure is more ordered than the structure adopted by free

water molecules and is energetically less favorable. In an aqueous solution,

the total hydrophobic surface area will be minimized if the hydrophobic

entities present in the solution come together. Minimizing the hydrophobic

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17

area minimizes the number of water molecules needed to encapsulate it,

which in turn stabilizes the system energetically9.

2.4 Basic structure of phospholipid membranes

Phospholipids in biological membranes are amphiphatic. They consist of a

hydrophobic hydrocarbon tail and a hydrophilic phosphate-containing

headgroup. As mentioned, the hydrophobic effect will cause them to form

bilayers, consisting of two sheets of lipids with their hydrophobic tails facing

each other, see figure 3. The hydrophilic headgroups shield the tails, forming

an interface between the hydrophobic core of the membrane and the aqueous

surroundings10

. There is no sharp border between the hydrophobic core and

the surrounding water as this interface region provides a zone of gradually

changing hydrophobicity, see figure 3. In 1992, Wiener and White

determined the structure of a bilayer of pure DOPC lipids11

. The

hydrophobic core was about 30 Å thick, while the interface region extended

about 15 Å on either side, see figure 3. The tails of the lipids can be of

different lengths and stiffnesses and the size and electrical charge of the

headgroup can differ. Steroidic lipids like cholesterol are very rigid and

confer stability9. The features and properties of a biological membrane,

thickness, fluidity, curvature, pressure, etc, will vary with the lipid

composition12

. It varies from organism to organism and between organelles

Figure 2. (A) A water molecule is dipolar with partial positive charges on the hydrogen atoms

(δ+) and a partial negative charge on the oxygen atom (δ-). The dashed lines represent the

nonbonding electron orbitals. (B) Each water molecule can form a maximum of four

hydrogen bonds. (C) A lipid in water. When confronted with a non-polar entity, like the

hydrophobic tail of a phospholipid, water molecules form a cage-like structure around it.

Water molecules not forming the “cage” have been omitted.

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18 Chapter 2 • Biological membranes

in the same cell, even between leaflets of the same bilayer. It is worth to

mention that Archaea, often found in extreme habitats, employ lipids that

differ substantially from the bacterial and eukaryotic ones, even though the

hydrophilic part of the head groups can be the same13

.

Figure 3. (A) A ribbon representation of a phosphatidylcholine lipid with a C17- and a C14-

hydrocarbon tail. Cyan, carbon; red, oxygen; blue, nitrogen; yellow, phosphate. Hydrogens

are omitted for clarity. (B) A simulated phospholipid bilayer. Molecular groups colored as in

A. Without (left) and with (right) surrounding water molecules. (C) The structure of a DOPC-

bilayer along the membrane normal, shown as the probability of finding different chemical

groups at a given distance from the center of the hydrocarbon core. Adapted from White and

Wimley (1999)14.

2.5 Biological membranes consist of more than lipids

One of the earliest models of a cell membrane depicted it as an ocean of

lipids with proteins floating around in it15

. The truth has been shown to be

somewhat different16; 17

. In some membranes, the lipids act more as a

cohesive between proteins with up to 30 000 molecules of integrated

membrane proteins per µm2 membrane

18; 19

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19

Chapter 3

Proteins in general

Out of 20 basic amino acids, a plethora of different structures can be built.

The human genome is at the moment estimated to contain 20,000 to 25,000

genes encoding proteins20

. Considering alternative splicing events and post-

translational modifications, the number of proteins with different chemical

composition is estimated to be more than fifty times higher21

.

3.1 Protein structure and folding in general

Proteins are heteropolymers of amino acids, with the amino acids linked in a

chain by peptide bonds. The amino acid side chains, or residues, protrude

from the chain and vary in structure, size and electric charge. For a protein to

be functional, its polypeptide chain needs to be properly folded into its

native conformation. Most of the information on how to reach the native fold

is present in the amino acid sequence itself, in the nature of the side chains

and the order in which they are linked22

. To quote Anfinsen from his Nobel

laureate lecture in 1972 on the folding of proteins: "The native conformation

is determined by the totality of interatomic interactions and hence by the

amino acid sequence, in a given environment." In an aqueous environment

like the cytoplasm, the hydrophobic effect will cause hydrophilic residues

and the polar peptide backbone to be hydrated by water molecules while

hydrophobic parts are hidden in the protein interior. In the hydrophobic core

of the membrane the situation is the opposite and the presence of hydrophilic

residues and the backbone will be energetically unfavorable.

Protein structure is often discussed in terms of different levels of order:

1. Primary structure, the sequence of amino acids.

2. Secondary structure, local folding of the chain. The most widely

occuring secondary structures of regular patterns are α–helices and

β-sheets, see figure 4.

3. Tertiary structure, the final folding of the chain, its spatial

arrangements of and interactions between elements of secondary

structure, see figure 4.

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20 Chapter 3 • Proteins in general

4. Quaternary structure, interactions between and arrangement of two

or more folded chains, or subunits, into a larger functional protein

complex, see figure 5.

Local interactions form first and a polypeptide can follow a large number of

different folding paths to reach the most energetically favored structure.

There exist no more than one or possibly a few native conformations for any

given naturally occurring protein23

.

Figure 4. (A) A polypeptide of five residues, side chains denoted by R1-5. The polar nature is

shown for one of the peptide bonds (highlighted in grey). (B) An α –helix is stabilized by

hydrogen bonds between residues i, i+4. Each helical turn rises 5.4 Å. (C) A protein with a

mainly helical tertiary structure, its two β–sheets indicated by an arrow. Cartoon

representation. (D) The same protein from the same viewing angle, surface representation.

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21

3.2 More on the quartenary structure of proteins,

protein-protein interactions

“A functional protein within the cell” most often refers to a complex of

interacting individual protein chains. The composition of protein complexes

can vary depending on what is needed for the moment in terms of cellular

architecture, regulation, metabolism, and signaling. Many proteins are part

of larger networks of protein-protein interactions in the cell. Some of the

proteins have high connectivity, interacting with a large number of different

partners. These “hub proteins” are often important for cell survival24

. Some

examples of protein complexes that are relevant for this thesis: (1) The Sec

translocon, which will be discussed to some length in chapter 5. The core of

this protein is a heterotrimer that can combine with different interaction

partners to translocate varying substrates across the membrane or inserting

them into it, see figure 5A. (2) The ribosome, the molecular machine

synthezising all proteins, consists of two subunits. In Archaea, the subunits

comprise in total about 50 relatively small protein chains and three RNA

chains, see figure 5B. (3) Chaperones are proteins involved in more transient

interactions, preventing misfolding events by binding to unfolded or

misfolded proteins until these can fold properly. However, the interaction

conveys neither information nor functionality to the interactants as a group

and the chaperone and its substrate are not considered forming a relevant

quarternary structure.

Figure 5. Examples of

protein complexes. Cartoon

representation, each subunit

is colored differently. (A)

The SecYEβ complex of the

Sec translocon. PDB file

1RH5. (B) The largest (50S)

of the two subunits of a

prokaryotic ribosome. The

orange bands represent

ribosomal RNA. PDB file

2X9S.

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22 Chapter 3 • Proteins in general

3.3 Evolutionary evolved functional substructures, protein

domains

Structure gives function. A protein, even a monomeric one, that performs

several tasks like binding a ligand, hydrolyzing ATP, etc, usually uses a

distinct substructure for each task. More often than not, these substructures

are autonomously folding units, called domains, indicating modularity

among protein folds. Indeed, it has been shown that during evolution,

proteins often have evolved by means of deletion, duplication or addition of

domains on the genetic level25; 26

. It has been estimated that out of a total

number of at least 10 000 observed folds, 80% of all protein sequences will

fold into one of 400 common folds, emphasizing how protein domains have

been reused during evolution27

. In paper IV, protein interactions are partly

discussed in terms of interacting domains.

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23

Chapter 4

Membrane proteins, what do they look like?

There are peripheral and integral membrane proteins. The peripheral ones

are loosely attached to lipids or proteins in the membrane via electrostatic or

van der Waals interactions, or covalently linked to a lipid anchor28

. The

integral ones traverse the membrane and experience the varying milieus of

the hydrophobic core, the interface region and the aqueous surroundings

outside of the bilayer. Integral

membrane proteins minimize

the cost of harboring the polar

polypeptide backbone within

the membrane by engaging its

polar groups in hydrogen

bonds. The two major

structural classes of membrane

proteins present two different

ways of doing this. (1) β-barrel

membrane proteins form

cylinders of amphiphatic β-

strands and (2) α-helical

membrane proteins span the

membrane with one or more α-

helices. See figure 6. The β-

barrels occur in the outer membrane of some bacteria and in the eukaryotic

organelles of bacterial origin, mitochondria and chloroplasts28

. Two to three

percent of the genes in Gram-positive bacteria have been estimated to

encode β-barrels29

. In comparison, about 25 % of the open reading frames in

most fully sequenced genomes have been estimated to encode α –helical

membrane proteins30

. From here on “membrane proteins” (MPs) refer to

integral α-helical membrane proteins.

Figure 6. The two major structural types of

membrane proteins, cartoon representation. (A)

Helical bundle. Bacteriorhodopsin, PDB code

1NTU. (B) β-barrel. Phosphoporin, PDB code

1PHO.

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24 Chapter 4 • Membrane proteins, what do they look like?

4.1 Structure and topology of α-helical membrane proteins

In addition to the four levels of structure, membrane proteins can be

described by their topology, what segments that are transmembrane and

extramembrane, cytosolic or extracytosolic (lumenal, periplasmic, etc.)

Transmembrane helices are described according to their orientation, whether

their N-terminal is cytosolic (Nin) or extracytosolic (Nout). Extramembrane

segments, loops, are simply described as cytosolic or extracytosolic.

4.1.1 The primary structure of a transmembrane helix is

adapted to its surroundings

With respect to the different

milieus a transmembrane helix

traverses, it is not surprising that

there is a statistical difference in

distribution of the amino acids in

different parts of the helix, see

figure 710; 31; 32

. The majority of side

chains found within the core of the

membrane are hydrophobic.

However, as more 3D-structures

are obtained, many examples of

charged residues and sequences

causing irregular secondary

structure within the hydrophobic

core have been observed 33

.

Charged and polar residues are better tolerated at the helix termini due to

snorkeling

Charged or polar amino acids are energetically better tolerated towards the

termini 31; 34

. Their side chains will “snorkel”, orienting themselves so that

they approach the interfacial, aqueous regions 35; 36

. This allows them to pull

hydrating waters into the hydrocarbon part of the bilayer and create polar

microenvironments for themselves, as seen in simulations 37

. Obviously a

long side chain is advantageous for snorkeling 37; 38

, see figure 8. However,

not all polar or charged residues in transmembrane segments are found in

positions allowing snorkeling.

Figure 7. Statistical distribution of amino

acids in different positions in a TMH.

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25

Figure 8. Snorkeling of side chains. Simulated model systems with the denoted amino acid

placed symmetrically with an offset of 5 positions from the center of the hydrocarbon core

(indicated by arrows). Note the bend induced by the shorter Asp side chains. Adapted from

Johansson and Lindahl (2006)37.

Tryptophans and tyrosines anchor themselves in the interface region

Tryptophan and tyrosine are especially favored in the interface region10; 39; 40;

41 and can virtually anchor a segment within the membrane

42. This rather

tight anchoring, employed to a higher degree by β-barrel proteins than by α–

helical MPs43

, seems to be due to both steric and electrochemical factors.

The large, flat and rigid ring structures of the Trp and Tyr side chains limit

access to the hydrocarbon core while the electric structure of the aromatic

rings favors interaction with the groups in the interface region. In addition,

both Trp and Tyr have one polar group each. Phenylalanine is also aromatic,

but entirely hydrophobic, and is not biased towards the interface region10; 40

.

4.1.2 Secondary structure in transmembrane segments can be

irregular

A coil is a segment where the peptide forms no regular secondary structure,

neither a helix nor a β-sheet, hence exposing its polar backbone, see figure 9.

Especially in channels and transporters, this feature occurs frequently and is

conserved and important for function44

. Coils are enriched in proline and

glycine residues. In TM helices, they especially confer a higher degree of

structural flexibility, creating swivels and hinges and also positioning side

chains properly for interactions45; 46

. Coils can be introduced into the lipid

bilayer as short kinks or longer breaks in the secondary structure of a

transmembrane helix, or as part of a reentrant loop or region (see the

following paragraph)10; 44

.

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26 Chapter 4 • Membrane proteins, what do they look like?

4.1.3 Other structural elements of helical membrane proteins

Reentrant regions are segments

that penetrate the lipid bilayer

without traversing it, entering

and exiting on the same side, see

figure 9. They have been

estimated to occur in at least

10% of all multispanning

membrane proteins, mostly in

ion and water channels47; 48

. As

defined by Viklund et al in 2006,

they can be (1) helix-turn-helix

and are, if long enough,

sometimes hard to distinguish

from two transmembrane

helices, (2) shorter segments of coil-helix or helix-coil and (3) even shorter

segments of purely irregular coil structure47; 48

. Reentrant loops are enriched

in prolines and small residues, especially glycines and alanines48

.

Polar backbone groups can be made available for hydrogen bonding by

other means than forming a coil. The π-helix bulge, or α-aneurysm, occurs

when a hydrogen bond is formed between residues in positions n, n+5

instead of the regular pattern of n→ n+4. This frees one carbonyl group

(C=O) for polar interactions. A tighter bond pattern of n→ n+3 can also

occur and the segment is then called 310-helix49; 50

. Interface helices are

found in the interface region, running roughly parallel to the membrane

surface. They are amphiphatic and on average nine residues long as reported

in 2005 by Granseth et al 10

.

4.2 Forces and motifs implied in the folding of helical

membrane proteins

The native fold of a membrane protein is mostly stabilized by interactions

between its TM helices. The current understanding of the factors stabilizing

a native fold is that van der Waals forces and hydrogen bonds dominate,

aided by the occasional salt bridge and aromatic interaction. Also, the lateral

pressure in the lipid bilayer, as well as the thickness of the bilayer, affects

protein stability.

Figure 9. Example of a reentrant region and

coiled segments within the membrane. The

membrane borders are shown as red and blue

lines.

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27

4.2.1 Intramolecular forces stabilizing the native fold

Van der Waals forces

It is still debated which the major factor that stabilizes membrane protein

folds is, but the total sum of the van der Waals interactions is a good

candidate 51

. Van der Waals forces occur between all atoms and molecules:

Non-polar molecules can become temporary dipoles if their total electron

density gets unequally distributed as the electrons move about. If close

enough, this dipole can induce a temporary dipole in the electron density of a

neighboring non-polar molecule, allowing electrostatic interaction. The

interaction gets stronger the closer the molecules are. The information on

how tight TM helices pack in the membrane is ambiguous 52; 53; 54; 55

, but the

residues in transmembrane regions do tend to be buried more often than

residues in soluble proteins or extramembrane regions56

, allowing a higher

number of van der Waals interactions if not necessarily stronger ones.

Hydrogen bonds

A hydrogen bond is the interaction between an atom with a non-bonded pair

of electrons (O, N, etc) and a hydrogen atom covalently bonded to a strongly

electronegative atom (N, F, O). The electronegative atom attracts the

electron density of the small hydrogen atom, decentralizing it and leaving

the hydrogen with a substantial positive partial charge57

. Carbon can also

take the place of the electronegative atom, resulting in a weaker hydrogen

bond. In membrane proteins, the TMH backbones can be involved in such

weak hydrogen bonds, CαH∙∙∙O 58; 59

. Even though the stabilizing effect of the

weaker bonds has been disputed 60; 61; 62

, it is likely to be substantial for

helices at close distances 60; 63

64

. Strong hydrogen bonds involving Asn, Asp,

Gln and Glu have been shown to drive helix oligomerization 65; 66; 67; 68; 69

.

The strength of any type of hydrogen bond depends on the distance, the

chemistry and relative arrangement of the involved atoms, and the nature of

the surrounding milieu 70; 71

. Even small structural changes can rather easily

break a hydrogen bond 62

.

Motifs allowing tighter packing of helices, thereby increasing stabilization

A number of motifs involving small side chains allow tight packing of TM

helices. The small residues are spaced in such a way that their side chains

end up on the same side of the helix, either i, i+4 or i, i+7, creating a surface

that allows close proximity. Statistics show that Glycine zippers like GxxxG,

(G, A, S)xxxGxxxG and GxxxGxxx(G, S, T) occur in more than 10 percent

of all known MP structures72. These motifs usually form right-handed

crossings of the helices. The motif GxxxG has been extensively studied 72; 73;

74; 75 and can readily, depending on sequence context, induce dimerization of

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28 Chapter 4 • Membrane proteins, what do they look like?

TM helices76; 77

. However, the majority of helix-helix interaction interfaces

seem to be formed by small residues in a i, i+7 pattern 76

, resulting in left-

handed crossing at small angles and knobs-into-holes packing, where side

chains i and i+3 are interdigitated between four side chains of the other TMH 78

. Tightly packed helices are stabilized by stronger van der Waals

interactions due to closer proximity of the interacting helices. Many helix-

helix interfaces are also stabilized by hydrogen bonds, both “classical” bonds

and the weaker bonds involving the peptide backbone, Cα-H∙∙∙O 64; 79; 80

.

Hildebrand et al have shown that the residues participating in hydrogen

bonds are more conserved than other buried residues 64; 79; 80

, and that the

packing preferences differ between channels and transporters and other

membrane proteins, mostly because these classes of protein are enriched in

different amino acids. In channels and transporters, the helix-helix interfaces

are not as tightly packed. Also, hydrogen bonds occur more frequently and

are often found in proximity to water-filled cavities providing alternative

bonding partners, allowing weaker bonds suitable for the structural

rearrangements needed for functionality 64; 79; 80

.

Aromatic residues can drive helix dimerization

A recent study by Sal-Man et al showed that the motif aromatic-xx-aromatic

is overrepresented in bacteria and that the motifs (Q, W, Y)-xx-(Q, W, Y)

could drive self-oligomerization of TMHs in a model system. The

oligomerization effect was stronger if the aromatic residues were close to the

interface region (see paragraph 4.1.)81

. Another study showed that

tryptophans in various positions promote homo-oligomerization and ruled

out that it was due to anchoring effects 82

.

Ion pairing/salt bridges

Salt bridges are electrostatic interactions between ions of opposite charge.

Within the hydrophobic milieu of the lipid bilayer, the di-electric constant is

very low and the interaction of a salt bridge is therefore strong. As briefly

mentioned in paragraph 4.1.1, charged residues (protonated Lys, Arg, His

and deprotonated Asp, Glu) are not common in transmembrane protein

segments. However, intramembrane salt bridges have been observed and are

of both structural and functional importance for their proteins 83; 84; 85

.

4.2.2 Lipid-protein interactions affecting the folded protein

The lipid composition of the bilayer affects the conformation of membrane

proteins in more ways than one.

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29

Lateral pressure in the membrane

Lipid molecules have different geometries, depending on the size of the head

group and how saturated their hydrocarbon tails are. Simply put, lipids

shaped as cylinders are bilayer-forming and lipids shaped like cones

introduce curvature 86

. If non-bilayer lipids are forced into the bilayer a stress

is introduced in the membrane. The stress is also affected by the charge

states of the lipid headgroup87

. The lateral pressure profile through the

membrane, or membrane curvature, differs with lipid composition, and

affects the folding and insertion of some proteins 88

.

Hydrophobic mismatch between membrane and protein

The lengths of the lipid hydrocarbon tails and the TM segments of

membrane proteins are not always matched. To compensate a mismatch,

long TM segments either tilt or distort via coils, or the surrounding lipid tails

extend. The tails of lipids surrounding a short TM segment compensate by

compression 89; 90; 91

. Mismatch has been shown to affect the functionality of

several proteins 92; 93; 94; 95; 96; 97; 98

, partly probably due to effects on helix

dimerization99

. Eukaryotic cells have also been suggested to utilize

hydrophobic mismatch to sort membrane proteins in the exocytic pathway 100; 101; 102; 103

.

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31

Chapter 5

Biogenesis and bilayer integration of α-

helical membrane proteins

I have discussed what membrane proteins look like and how their final

structure is stabilized. But how does it all begin, how are they integrated into

the membrane? The vast majority of proteins are synthesized in the cytosol,

where the ribosomes translate mRNA codons into amino acids and add them

to the growing polypeptide chain. Soluble cytosolic proteins simply fold as

they emerge from the ribosome. For proteins meant for secretion or

integration into a lipid bilayer, the process is more complicated and there are

both co-translational and post-translational pathways for translocation across

or insertion into the membrane.

The most primary and well-studied system is the SRP-dependant Sec

pathway. Its major components are the signal recognition particle (SRP), the

signal recognition particle receptor (SR) and the core of the secretase (Sec)

translocon, which forms a pore through the membrane (reviewed in 14; 104

).

This system is present in all organisms studied so far105

.

After translation is initiated, there are two major steps in membrane

protein biogenesis, see figure 10:

1. Targeting to the Sec translocon. The SRP will recognize and bind a highly

hydrophobic stretch of the nascent polypeptide chain, the signal peptide,

when it emerges from the ribosome. In eukaryotes, but not in

prokaryotes, this arrests the chain elongation until the ribosome contacts

the translocon. SRP then binds to its receptor in the ER membrane

(eukaryotes) or in the plasma membrane (prokaryotes), which catalyses

the transfer of the ribosome-nascent chain-complex to the translocon106;

107.

2. Translocation/integration. The exit tunnel in the ribosome is aligned with

the translocon pore108

. When the signal peptide reaches the translocon it

either reorients, placing the N-terminal in the cytosol, giving the protein

an Nin-orientation, or it does not, resulting in an Nout-orientation. As

translation and chain elongation proceeds, the nascent chain passes

through the translocon channel. Segments that are of sufficient length and

hydrophobicity will enter into the lipid bilayer109

.

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32 Chapter 5 • Biogenesis and bilayer integration of membrane proteins

Figure 10. SRP-dependant co-translational insertion of an ER membrane protein. Adapted

from Rapoport (2007)14.

5.1 The Sec translocon channel

The Sec core translocon is a heterotrimer in all organisms studied, two of its

subunits are highly conserved and essential while the third is not105

. It is

denoted Sec61αβγ in eukaryotes, SecYEG in eubacteria and SecYEβ in

Archaea. The major component is the pore protein, subunit α/Y. In 2004, a

leap in the study of membrane protein integration was made when van den

Berg et al presented the high-resolution three-dimensional structure of a

closed SecYEβ translocon channel110

, see figure 11. Some of the most

interesting features of the pore is (1) a proposed lateral gate, where the pore

opens like a clamshell towards the bilayer, exposing the translocating peptide

to the lipids and allowing it to partition between the aqueous channel and the

bilayer110; 111; 112; 113

. (2) A plug that closes the non-cytosolic end and prevents

translocation110; 114

. (3) A ring of hydrophobic residues in the middle of the

pore, shaping it into an hourglass and further sealing it so there can be no ion

leakage across the membrane. In the open state the ring most likely prevents

unspecific interactions between the channel and the translocating protein 112;

115; 116; 117. The active translocon has been observed to be a multimer of Sec

heterotrimers and other proteins, but the actual number of subunits and their

arrangement are controversial118; 119; 120

. During translocation/insertion, the

pore dimensions seem to adjust to the nascent protein chain121

. An elongating

hydrophobic segment is thought to bind to a specific site, wedging into the

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33

gate and inducing structural changes that eventually will displace the plug

and open the gate 110; 122; 123; 124

.

Figure 11. The SecYEβ translocon pore .(A) Top view from the cytosolic side. (B) Side view.

SecE and Secβ are shown in black, SecY in grey. The lateral gate in SecY is shown in orange

and the pore plug and ring in red. PDB code 1RH5.

5.2 The versatile translocon

So, the Sec heterotrimer, or a multimer thereof, forms the primary translocon

channel. By combining with different proteins responsible for transfer of

peptides to the channel, chaperone function and movement through the pore,

the translocon translocates/integrates substrates of varying characteristics.

Other proteins implied in the SRP-dependant pathway

In bacteria, the YidC protein is needed for the co-translational integration of

many membrane proteins125; 126; 127; 128

. In eukaryotes, the monomeric TRAM

(translocation-associated membrane protein) appears to fulfill a similar

function129; 130; 131; 132

. Also, the tetrameric translocon-associated protein

complex, TRAPαβγδ 133

, has been shown to be essential for the binding of

some signal sequences to the eukaryotic translocon134

. (Note that although

the names are confusingly similar, TRAM and TRAP are two completely

separate proteins.) Cross-linking experiments indicate that these proteins

contact the nascent peptide as it emerges from the translocon, facilitating

integration into the bilayer128; 129; 135; 136

. In 2005, Ménétret et al captured the

structure of a mammalian translocon-ribosome complex at low resolution.

Their data suggests a complex of one ribosome, four Sec heterotrimers and

two TRAP molecules118

. Recently, the cytosolic protein SYD (Suppressor of

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34 Chapter 5 • Biogenesis and bilayer integration of membrane proteins

SecY Dominance) was suggested to recognize misassembled SecY channels

in gram negative bacteria, acting as a quality control system137

.

Post-translational translocation

The Sec translocon is also involved in post-translational translocation of

proteins. After synthesis, aggregation is prevented by cytosolic chaperones

binding to the protein before it is transferred to the translocation machinery.

In eubacteria like E. coli, the translocating protein is pushed through the pore

in an ATP-demanding process by the cytosolic SecA protein instead of the

ribosome. A complex of SecD, SecF and YajC also participates in the

process138; 139

. SecA is also needed for the translocation of the extracellular

domains of several membrane proteins utilizing the co-translational

pathway140. In yeast, and most likely all eukaryotes, the translocating protein

instead moves through the pore by Brownian motion after the signal

sequence binds to the translocon. The luminal chaperone and ATPase BiP

binds the protein and prevents movement back into the cytosol. This process

also requires the additional transmembrane Sec62/63 complex.

Modifying proteins

Signal peptidase141

will cut off the signal peptide and release proteins for

secretion into the exoplasm. For MPs, the signal sequence is either cleaved

off or integrated into the membrane as a TMH. In eukaryotes, a modifying

enzyme attaching N-linked glycans to the protein is also found in the close

vicinity of the translocon (see section 7.3.1).

5.3 Other systems for insertion of membrane proteins

Even though most membrane proteins seem to be inserted via the SRP-

dependant Sec pathway, there are other insertion mechanisms. These are

found in the bacterial membranes and in the membranes of the eukaryotic

mitochondria, chloroplasts and peroxisomes. A very brief review of these

systems will be given here.

Mitochondria lack the Sec-translocon completely. Instead this dual-

membrane organelle is equipped with an elaborate system for import and

insertion of membrane proteins, involving six major protein complexes and a

number of chaperones142; 143

. Of these proteins, the Oxa1 protein has

homologues in bacteria, some Archaea and chloroplasts125; 144

. As mentioned,

the homologue in E.coli, YidC, assists the SecYEG translocon in insertion

of some proteins into the cell membrane125; 126; 128; 144

and has recently been

shown to act as a translocon on its own125; 145; 146

. An interesting case has

been reported where the N-terminal of a E.coli protein is inserted via the

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35

YidC only pathway and the C-terminal subsequently is inserted via

SecYEG/YidC147

.

In peroxisomes, some membrane proteins are imported co-translationally

in a not yet completely elucidated process while others come in vesicles

budded off from the ER148; 149

. Chloroplasts have translocons of the inner and

outer membranes (TOC and TIC) as well as the twin arginine translocon

(Tat) that translocates folded proteins across the thylakoid membrane and

seems to be involved in the insertion of some integral membrane proteins150;

151; 152; 153. Tat is also found in Bacteria, Archaea and plant mitochondria

154.

From here on, “translocon” will refer to the Sec-translocon as engaged in the

SRP-dependant pathway.

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37

Chapter 6

How is topology decided during biogenesis

and folding?

Deciphering the topology signals and folding of a membrane protein are

often described as separate processes in a simplified two-step model155; 156

. In

this model, topology is established first, as the helices are thought to insert

individually and to be stable on their own in the membrane. Thereafter the

helices are envisioned to interact with each other within the membrane and

form the final tertiary structure. Although this model may be true for some

proteins156

, it is too simplified for others. There are several examples on

helices of low hydrophobicity, marginally hydrophobic helices, which are

not inserting well on their own, but are transmembrane in the folded

protein44; 157; 158; 159; 160; 161

. During the folding of aquaporin1, the prospective

TMH2 and TMH4 are even extramembrane after passing the translocon and

are pulled into the membrane as TMH3 later flips 180 degrees, assisted by

the nascent C-terminal helix bundle120

. In addition, it is known that some TM

helices interact transiently with proteins associated with the pore upon

entering into the lipid bilayer (see section 5.2). Emerging evidence also show

that helices can stay in close proximity to the translocon until stable

interactions can be formed with subsequent helices162

and that some even can

be retained within the channel due to specific interactions163

.

Still, the three major factors affecting how a peptide chain is threaded

through the membrane seem to be hydrophobicity (will a segment partition

into the bilayer?), length of the hydrophobic segment (can it span the

membrane?) and the presence of charged residues in the loops (will a

segment be retained in the cytosol?). For some segments, the signals to adopt

a certain topology can be so strong that they force an intermediate segment

to become transmembrane, despite low hydrophobicity160

. Timing of the

elongation and insertion can also affect topology, as the proper segments

need to be available at the correct moment, and so on164

.

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38 Chapter 6 • How is topology decided?

6.1 Charged residues in loops, the positive inside rule

Positively charged, basic residues are strong topologenic signals with a

preference for the cytosol. A simple way to predict the topology of a

membrane protein is to localize the most hydrophobic parts (putative TMHs)

and then summarize the number of positively charged residues in the loops

on either side of the membrane. The side sporting the highest number of

positively charged residues is likely to face the cytosol165

. This effect is

called the positive inside rule and has been shown to hold for most

organisms41; 165; 166; 167

. Acidic residues do not effect topology as strongly and

show no statistical preference for loops on either side of the membrane 41; 168

.

However, they seem to have a greater influence on topology in Sec-

independent translocation169

.

Effect of cytosolic basic residues on the insertion of TM segments

If placed downstream of and close to a transmembrane segment of Cin-

orientation, a single Arg or Lys can lower the apparent free energy of

insertion of that segment with ~0.5 kcal/mol 170

.The effect is additive, the

more residues of positive charge, the lower the free energy. Also, a stretch of

six lysines has been shown to affect insertion from a distance of up to ~25

residues away for a single-spanning protein and up to ~12 residues away for

a TMH in a multispanning protein, when expressed in an in vitro system170;

171. Histidines are not charged at physiological pH, but have a similar effect

if the pH is lowered168

.

Possible mechanisms underlying the positive inside rule

The exact mechanisms behind the positive inside rule are not yet fully

understood, and they may differ slightly between Archaea, Bacteria and

Eukarya. For example, the retaining effect of positive charges is more

pronounced in E.coli than in microsomes (miniature eukaryotic ER

membrane)172

, most likely due to the much stronger electrochemical

potential across the bacterial inner cell membrane. The ER membrane

potential seems to be non-existing. In E. coli, the dominance of positive

charges over negative charges as determinants of topology has been

proposed to be due to the lipid composition of the cell membrane. Several

E. coli proteins have been shown to be dependent on the presence of lipids

with a net zero charge to achieve the proper topology173; 174; 175

. It is thought

that these lipids dilute the otherwise strong negative charge of the lipid head

groups so that the acidic residues (aspartic and glutamic acid) stay

protonized and thus uncharged176

. Specific interactions with the translocon

have been reported to contribute to the orientation of the first TMH, the

signal sequence177; 178

. Also, electrostatic interactions between anionic

lipids and positively charged residues have been proposed to help retaining

positive charges in the cytosol179; 180; 181; 182

.

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39

6.2 Hydrophobicity as recognized by the translocon

Traditionally, the hydro-

phobicity of an amino

acid has been

determined by its ability

to partition between water

and an organic solvent

(See 183

for benchmarking

of experimental and

statistical hydrophobicity

scales). A more recent

method includes the

energetically important

effect of the protein

backbone on insertion39;

184; 185. There is also a

biological hydrophobicity

scale available, the Hessa scale186

. Rather than looking at the hydrophobicity

of the individual amino acids or side chains, the Hessa scale gives

information on how each side chain will affect the recognition of a helix as

transmembrane by the translocon. See figure 12. ΔGapp ≤ 0 kcal/mol means

that the amino acid favors recognition by the translocon. Positional effects,

as discussed in paragraph 4.1.1, are also taken into consideration. For the

work presented in this thesis, the Hessa scale and tools for predicting the

change in free energy upon insertion based on this scale were employed.

6.3 Marginally hydrophobic transmembrane helices do

exist

A number of TM segments have been observed in experiments where they

did not insert well into the membrane without the presence of other TM

segments120; 159; 187; 188

. Also, as discussed in paper I, the primary sequences of

many TM helices seen in three-dimensional structures do not indicate them

to be transmembrane. For instance, (LacY) of E. coli189

and the glutamate

transporter homologue of P. horikoshii (GltPh)44; 190; 191

both contain several

helices of low hydrophobicity, see figure 13. We take a closer look at GltPh in

paper II, and LacY is examined in paper III

As with coils, many marginally hydrophobic helices contain functionally

important residues, some of them responsible for interactions with other

subunits. However, such interactions are not likely to form at the stage of

integration into the membrane.

Lundin et al showed recently that two Asn- or Asp-mediated hydrogen

Figure 12. The Hessa scale. Apparent change in free

energy (ΔGapp upon translocon-mediated insertion

into the ER membrane. Adapted from Hessa et al

(2007) [1].

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40 Chapter 6 • How is topology decided?

bonds to a well-integrating TMH can improve the insertion of a marginally

hydrophobic one192

. In paper I, we investigate the effect of the sequence

context on the insertion of marginally hydrophobic helices. Ota et al

(1998)160

reported an example of well inserting segments with strong

topological signals forcing a less hydrophobic segment to insert into the

membrane, which sustains the findings we present in paper I. In paper II, we

present evidence indicating that some marginally hydrophobic TMHs are not

initially recognized as transmembrane and could be repositioned as a post-

translational event.

Figure 13. Marginally hydrophobic helices. (A) LacY, the lactose permease from Escherichia

coli. PDB code 2cfq. (B) GltPh, the glutamate transporter homologue from Pyrococcus

horikoshii. PDB code 2nwl. The TMHs are colored according to their predicted insertion

propensity, given as the apparent change in free energy upon integration into the membrane

(ΔG). A segment of ΔG≤0 has a reasonable insertion propensity, while a segment of ΔG≥0

does not. Prediction of hydrophobicity was carried out with the ΔG predictor, see section 7.2.

Positively charged residues (Lys, Arg) are marked with circles in light pink and negatively

charged residues (Asp, Glu) are marked with circles in cyan. Borders of the membrane are

indicated by red and blue lines.

6.4 Length of the hydrophobic segment

A transmembrane segment has to span the bilayer. A perfect α-helix raises

5.4 Å per turn, each turn employing 3.6 amino acids. To span the

hydrophobic part of the bilayer, which is about 30 Å wide, a segment of

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41

about 20 residues is needed. However, it has been reported that helices of 12-

40 residues can be accommodated in biological membranes10; 38; 193; 194; 195

.

The short helices need to be highly hydrophobic31

and the membrane bilayer

seems to distort to harbor these segments The longer helices can either be

slightly distorted, tilted relative to the normal of the membrane196

or force

the lipids to stretch, introducing another kind of lipid mismatch than the

short segments90

. (See also section 4.2.2.)

6.5 Reentrant loops

The topological signals resulting in reentrant loops are not yet well

understood and may very well vary depending on the nature and length of

the segment. As we propose in paper II for the ClcA H+/Cl

− exchange

transporter of E. coli197

, long reentrant loops à helix-turn-helix are likely to

insert as hairpins and then reposition themselves relative to the membrane

during later folding events. Detailed biochemical studies from the laboratory

of Sakaguchi have presented examples of both co- and post-translational

integration of reentrant loops198; 199

.

6.6 Other factors

Helices interacting with each other before entering the lipid bilayer can

“hide” charged or polar residues in interhelical hydrogen bonds69

,

increasing their apparent hydrophobicity. Also intrahelical ionic bonds,

between residues on the same side of a helix, have been shown to improve

insertion 200

. Regardless of charges, just the plain presence of other

polypeptide entities can effect insertion in a favorable way158; 162; 201

. This has

been explained by the greater capacity of polypeptides to retain hydration

water201

. Another factor affecting insertion may be when the α–helical

structure is formed. It has been shown that α –helices can form already in the

ribosomal exit tunnel202

. However, segments of low helix-forming propensity

may not become helical until a very late state during the integration into the

lipid bilayer 203

.

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43

Chapter 7

Structure analysis

The function of a protein depends on its structure and folding. The three-

dimensional structures of proteins are thus of uttermost interest. For

example, once transmembrane protein segments were thought to be perfect

α-helices, long enough to span the lipid bilayer. As more 3D-structures of

MPs have been obtained, it has become clear that TM elements of higher

complexity are commonly occurring, including the previously described

coils and marginally hydrophobic TMHs. As discussed earlier, these

elements are generally important for the function of the protein. This

knowledge allows us to better understand the mechanisms behind protein

functionality and the effect of the primary sequence on folding. For the final

structure of membrane proteins, topology is a vital part. There are three ways

to attain information on topology: (1) Solve the three-dimensional structure.

This is especially challenging for membrane proteins, but gives detailed

information on all aspects of the structure, not only the topology. (2)

Investigate the topology by biochemical means (see section 7.3). (3) Predict

the topology of a protein of interest based on previous knowledge. A number

of in silico topology predictors have been developed as the collective dataset

has grown during the last decades30; 204; 205; 206; 207; 208

. However, so far there

are no tools integrating predictions of topology, coils, helix packing8; 209

,

surface accessibility, and so on, into a detailed prediction of membrane

protein structure. Many more analyses of membrane protein structures are

needed.

7.1 Obtaining three-dimensional structures

The most commonly used techniques for solving three-dimensional

structures are X-ray crystallography210; 211; 212

and nuclear magnetic

resonance (NMR) 213; 214; 215

, both allowing resolution down to the atomic

level (about 1.5 Å or lower). Electron microscopy (EM) gives images of

lower resolution, revealing the general shape of a protein. Due to their poor

solubility and dependence on the lipid bilayer for proper structure,

membrane proteins are not easy targets. However, the techniques for

purification and preparation of the targets, as well as the visualization

techniques themselves, are constantly under development, and the number of

solved structures of membrane proteins has increased steadily since the first

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44 Chapter 7 • Structure analysis

high-resolution structure was solved in 1985216

. Nonetheless, the number of

solved structures for soluble proteins is by far higher. In July 2010, there

were 61,860 structures of proteins in the protein data bank (PDB)217

, 4920 of

these turned up in a search on the word “membrane” and 2205 in a search on

the word “transmembrane”. The orientation of membrane proteins (OPM)

database218

includes “all unique experimental structures of transmembrane

proteins and some peripheral proteins and membrane-active peptides” found

in PDB. In July 2010, OPM included 925 structures.

7.2 In silico investigations

A number of predictors and simulation methods are available. I will only

address the in silico tools that I’ve used in my research.

Predicting insertion propensity

To predict insertion propensity of a segment, either computational

simulations can be performed or knowledge gathered experimentally can be

applied.

As mentioned earlier, the study by Hessa et al31

gathered information on

how hydrophobicity, segment length and position of each amino acid in the

segment affect the insertion propensity. The authors incorporated their data

into equation 1 and later on in the “ΔG predictor” available at

http://dgpred.cbr.su.se/, a tool for calculating the ΔG for a specific segment

or for finding potential TM segments within a longer sequence.

Equation 1. The first term sums the individual, position-dependant (i) contributions for each

amino acid (aa) in the sequence. The second term takes the helical dipole moment into

consideration. The last term was fitted and b1, b2, and b3 were optimized to account for effects

due to length (l), based on the data from the study of Hessa et al. See 31 for details.

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45

A Monte Carlo-based simulation can give information on both insertion and

peptide interactions

Monte Carlo methods are especially useful in studying systems with a large

number of coupled degrees of freedom, such as a system containing peptides

and a membrane. In a Monte Carlo-based simulation, a number of

computational steps are carried out, each step performing random changes of

the parameters of interest (here the conformation and location of the

polypeptide(s)). The change resulting in the most stable system, the system

of the lowest all-atom potential energy, is selected and the process is iterated

a set number of times. To appreciate the propensity of a polypeptide to insert

into the membrane, several runs are performed, starting with the peptide(s)

in different (random) positions. The fraction of obtained low-energy states

where the peptide(s) end up traversing the membrane is then regarded as the

insertion propensity. If more than one peptide is included, possible

interactions between the peptides can also be evaluated219; 220

.

Prediction of protein topology

The earliest predictors of protein topology were based on (non-biological)

hydrophobicity plots221

, but performed poorly. This is perhaps not so

surprising considering the issues discussed in chapter six and section 5.2.

Later methods were mostly based on statistics, using experimentally derived

datasets to train computational models to recognize TMHs, interface regions,

etc30; 48; 222; 223

. When needed in our work, we used Scampi, a method

evaluating a primary protein sequence without previous training, employing

the biological Hessa hydrophobicity scale and the positive inside rule in a

simple model204

.

7.3 Determining membrane protein topology

experimentally

How can the topology of a membrane protein be determined? One way is to

utilize a modification that can occur in only one of the compartments

separated by the membrane, like glycosylation224

or cysteine labeling173; 225

.

By gradually introducing acceptor sites for this modification in different

positions and analyzing if the modification comes about or not, the topology

can be deciphered. Reporter proteins that will be active only on one side of

the membrane is a variation on the same theme, but can in general only give

information on the location of the protein termini. Another approach is

“membrane shaving”, where all extramembrane protein loops are cut into

pieces by a peptidase226; 227

. The individual peptides in the resulting mix are

then identified using mass spectrometry, which might require specific

modification of the peptides228

, depending on the protease used.

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46 Chapter 7 • Structure analysis

7.3.1 N-linked glycosylation as topology reporter and

molecular ruler

The enzyme Oligosaccharyl Transferase is found on the lumenal side of the

ER membrane. It recognizes the peptide sequence X1NX2(S/T)X3 and will

attach one sugar moiety to the asparagine-residue (N). The process is co-

translational229

. The attached oligosaccharide increases the molecular mass

of the protein with approximately 2 kDa230

, and glycosylated and

unglycosylated forms of the protein can easily be separated by SDS-PAGE.

In the recognition sequence, X1-3 cannot be Pro. NXT is preferred over NXS,

as the recognition of the latter sequon is more affected by the residues in

positions X1-3231; 232

.

The active site of the transferase is located at a fixed distance from the

membrane. Half-maximal glycosylation will be achieved if the acceptor site

is (a) 14 residues N-terminal of a transmembrane segment or (b) 10 residues

C-terminal of the most C-terminal TM segment. The position of a TM

segment can thus be determined by finding the “minimal glycosylation

distance” (MGD), the position where the acceptor-asparagine is glycosylated

at half-maximal efficiency233; 234; 235

.

7.4 Determining the propensity to integrate into the

membrane

The method used in this thesis to determine insertion propensity of a protein

segment employs N-linked glycosylation. A model protein carrying the

segment of interest and two glycosylation recognition sites is expressed in an

in vitro system. Unglycosylated, singly and doubly glycosylated protein

species are separated by SDS-PAGE and the signal intensities from the

bands on the gel are quantified. The insertion propensity is reported as the

fraction of inserted protein species and is determined by the glycosylation

status of the protein construct. The apparent change in Gibbs free energy

upon insertion (ΔGapp) can be calculated for this system, using a simplified

model where the translocon-mediated integration, the partioning of the

protein segment between the aqueous pore of the translocon and the

hydrophobic bilayer, is regarded purely as an equilibrium process31

. See

equations 2 and 3.

Where Keq is the equilibrium constant, finserted is the fraction of protein

species where the segment was inserted into the membrane, R is the gas

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47

constant and T is the absolute temperature.

7.4.1 Model proteins, LepI and LepII

As mentioned earlier, a highly hydrophobic signal peptide is required for a

nascent polypeptide to enter the translocon-mediated insertion pathway. To

be able to determine how well a less hydrophobic segment insert into the

membrane, either the entire native protein is used or the segment of interest

can be isolated by fusing it to a model protein. In this work, the model

proteins were two modified versions of the well-studied E. coli membrane

protein Leader peptidase B (Uniprot P00803), called LepI and LepII236

.

Leader peptidase B is a protein of 324 amino acid residues and two TMHs.

Both its short N-terminus and the large C-terminal domain face the lumen

when expressed in an in vitro system of rabbit reticulocyte lysate172; 234; 237

. In

LepI, the fused segment replaces a part of the C-terminal loop. In LepII it

replaces the second TMH. The N-terminal of the segment can thus be

positioned either in the lumen (Nout, in LepI) or in the cytosol (Nin, in LepII)

depending on which model protein that is used, see figure 14. Both model

proteins carry two glycosylation recognition sites, placed on appropriate

distances from the lipid bilayer in a manner such that the location of the

fused segment (inserted/not inserted) is mirrored by the glycosylation status

(see figure 14). An apparently systematic, yet unexplained, difference of

about 1 kcal/mol in free energy upon insertion between the two models has

been reported. That is, any segment is likely to insert more efficiently when

introduced into LepII compared to LepI236

.

To study segments containing several transmembrane parts, extra

glycosylation sites can be introduced by altering the segment sequence.

Figure 14. Lep model proteins. Grey rods (TMH1, TMH2), native transmembrane helices of

LepB. Black rod, segment to be investigated. G1, G2, G3, positions for glycosylation

recognition sites. The sites recieve a glycan (Y) if translocated across the membrane to the

lumenal side. A) Lep I. One glycosylation indicates insertion of the segment of interest. (B)

LepII. Two glycosylations indicate integration of the segment of interest into the membrane.

Note that the N-terminal af LepII is longer than for LepI, in order to ensure glycosylation of

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48 Chapter 7 • Structure analysis

site G3.( (C) Representative picture of a LepI-construct expressed in the presence (+) or

absence (-) of rough microsomes (RM). Unglycosylated (○), singly (●) and doubly (●●)

glycosylated forms are indicated. Mr, molecular ruler, protein sizes in kDa.

7.4.2 In vitro protein expression

Intact cells are not needed for protein expression. Cell lysates containing all

the necessary molecules are enough and facilitates labeling and purification.

During the last four decades a number of in vitro systems have been made

commercially available, prokaryotic as well as eukaryotic238

. They all

contain a transcription/translation system as well as the signal peptide

receptor system. When supplied with a polymerase of choice, some of these

systems can conveniently transcribe and translate a protein directly from a

DNA plasmid or even a PCR fragment under the control of the appropriate

promoter sequence. To facilitate a eukaryotic ribosome binding to the

mRNA and ensuring a high rate of translation, it is important to include the

Kozak consensus sequence239

upstream of the gene to be expressed.

In a lysate in vitro system, many proteins are already present. To facilitate

visualization of the newly synthesized proteins only, expression can be

carried out using radioactive amino acids, followed by proper visualization

methods. Radioactivity also facilitates quantification of the expressed

proteins.

To study the biogenesis of membrane proteins, the system is

supplemented with rough microsomes, small vesicular fragments of rough

ER.

7.5 Determining quartenary protein structure, the

composition of protein complexes

To fully understand the biological system that is a cell, a map of its protein-

protein interactions is needed. The last decade or so, a number of techniques

for deciphering how and when the proteins in a cell interact have been

developed. The major categories are complementation assays, affinity

purification in combination with mass spectrometry, and single molecule

techniques, as extensively reviewed by24; 240; 241; 242; 243

. Also, blue native

PAGE and clear native PAGE are two most useful techniques for

determining the composition of protein complexes244

245

. The latter three

methods are not suited for high-throughput work. During the latest decade,

several large scale high-throughput efforts employing complementation

assays and affinity purification have been carried out to map the protein

interactome, the set of all protein-protein interactions in a cell246; 247; 248; 249;

250; 251. Most were carried out in the yeast Saccharomyces cerevisiae, a

unicellular eukaryotic organism of about 6000 gene-encoded proteins252

. In

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49

paper IV, we present a comparison between datasets obtained with common

high throughput techniques and a collection of individual biochemical

studies.

Methods for elucidating protein interactions

In complementation assays, the direct pairwise interaction between two

proteins is investigated. The proteins are recombinantly fused to two halves

of a reporter protein and if they interact, the two halves will reconstitute the

functional reporter protein. In yeast, due to the possibility of mating, this

allows high-throughput screening. The original method is called yeast two

hybrid (Y2H)253

, where the reconstituted protein is a transcription factor that

initiates the synthesis of the actual reporter protein. This method does not

work well for membrane proteins, which cannot enter the nucleus. However,

there are modified versions where the reporter protein is fused to protein

fragments that upon complementation will cause the reporter to be released

from the protein complex243; 254

. There is also a fluorescent complementation

method, BiFC255

and fluorescent methods based on fluorescent/biolumi-

nescent resonance energy transfer240

.

In affinity purification, a protein of interest, the bait, is recombinantly

tagged with a protein segment with high affinity for a ligand. After in vivo

expression, the tag allows purification of the bait and, if the proper

purification conditions are chosen, its interaction partner(s). This method

allows detection of protein complexes, even subunits that do not interact

directly with the bait. However, the interactions have to be strong to survive

the purification procedure. In yeast, the most commonly used tag consists of

an IgG-binding peptide separated from a calmodulin binding peptide by a

recognition site for a protease. This double tag allows purification in two

steps, minimizing the number of false interaction partners. The procedure is

often referred to as TAP, tandem affinity purification246; 247; 248; 256; 257

.

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51

Chapter 8

Summary of the papers

8.1 Paper I - Membrane insertion of marginally

hydrophobic transmembrane helices depends on

sequence context

In this paper we studied helices that are not predicted to be sufficiently

hydrophobic to be recognized by the translocon, but still are transmembrane

in the obtained three-dimensional structure of the protein. We searched for

transmembrane segments that were marginally hydrophobic, with a predicted

change in free energy upon insertion of less than or equal to 1.4 kcal/mol,

corresponding to an insertion efficiency of 8% in our experimental model

system. To allow for some flexibility regarding correct placement with

regard to the membrane, five upstream and five downstream residues were

added to the segment reported to be transmembrane. The subsegment

predicted to be of the highest hydrophobicity within this expanded sequence,

but still marginally hydrophobic, was then considered in the experimental

investigations. Out of 819 transmembrane helices from known 3D-structures

of integral membrane proteins, 136 TMHs (or 17 %) were predicted to be of

such low hydrophobicity.

We screened 16 of the segments predicted to be marginally hydrophobic

and found that most of them were indeed not inserting efficiently when

isolated from their native protein. When we included the flanking segments

up to the neighboring helices or at maximum 20 residues, we found several

examples where the insertion improved, of which most could be explained

by the “positive inside”-rule. For five of the marginally hydrophobic helices

we also included well-inserting flanking segments consisting of either the N-

terminal neighboring helix, the C-terminal neighboring helix, or both these

helices. This improved the insertion of three of the five helices. Of the other

two, one is known not to be inserted by the translocon, but rather via a post-

translational structural rearrangement, and the other one has not been

reported before. Statistical analysis of a homology-reduced version of the

dataset, containing 388 TMHs from 102 proteins, showed that the number of

marginally hydrophobic helices increases with the total number of TMHs in

the protein, that they are less common as the first, N-terminal TMH, and that

they constitute a higher fraction of the helices in channels and transporters

than in other proteins. We could also see that marginally hydrophobic TMHs

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52 Chapter 8 • Summary of the papers

are more buried (less exposed to the lipid bilayer) than other TM helices.

However, buried helices were mostly observed in structures of protein

complexes, and were usually buried by other protein chains rather than via

intramolecular contacts, thus not making it likely to affect the initial

integration of the TMH into the membrane.

8.2 Paper II - Repositioning of transmembrane alpha-

helices during membrane protein folding

The glutamate transporter homologue from P. horikoshii, GltPh, has a

complicated three-dimensional structure with several marginally

hydrophobic transmembrane helices, some disrupted by coils within the lipid

core of the membrane. In this paper we showed that some of these segments

have overlapping, more hydrophobic regions that are more efficiently

recognized by the translocon. We could also establish that at least one of the

overlapping segments is initially integrated into the membrane rather than

the segment seen in the structure. In a non-redundant dataset of membrane

proteins of known three-dimensional structures, a substantial number of

transmembrane helices were predicted to be poorly recognized by the

translocon, but to have overlapping, better recognized segments. We

concluded that post-translational repositioning of TM helices in relation to

the membrane may not be uncommon during the folding of multispanning

membrane proteins.

8.3 Paper III - Insertion Properties of Marginally

Hydrophobic Helices in the LacY Lactose Permease

Transporter

The E. coli lactose permease LacY contains several marginally hydrophobic

TM helices. We investigated whether these helices, especially those in the C-

terminal end of the protein, initially integrate into the lipid bilayer as helical

hairpins. We combined molecular simulations based on the Monte Carlo-

method and the same in vitro, glycosylation based experimental setup as in

papers I and II. Surprisingly enough, our results indicate that the marginally

hydrophobic TM helices of the C-terminal half of LacY do not integrate into

the membrane as helical hairpins. This could imply that interactions need to

be established between several of the TMHs before they are collectively

released from the translocon machinery into the lipid bilayer.

.

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53

8.4 Paper IV - Quantitative assessment of the structural

bias in protein-protein interaction assays

The interchange of information and matter in a cell depends on interactions

between proteins. The last decade, a number of large-scale investigations of

the yeast interactome have been carried out, employing tandem affinity

purifications (TAP) coupled to mass-spectrometry, or yeast-two-hybrid

(Y2H) techniques. However, the overlap between the resulting datasets is

surprisingly small, indicating systematic differences in detection between the

experimental setups. In order to characterize the biases for each system, we

have analyzed two available TAP-datasets (Krogan248

, Gavin246

) and Y2H

data as collected from the IntAct database and compared them to manually

curated data collected from small-scale experiments.

Note that Y2H identifies direct pairwise interactions and is known to give

a high degree of false positives, while TAP, due to the purification steps,

identifies protein complexes with relatively strong interactions. For TAP, the

results to a higher degree depend on variations in handling, purification

protocol and choice of mass spectrometry (MS) method for identifying the

interactants. Neither method is suited for membrane proteins.

Previous studies have suggested disordered regions and repeating

domains to be important for protein-protein interactions. Certain non-

repeating protein domains have also been proposed to be especially

interactive. In addition, protein size and abundance are, even though not

completely orthogonal to the other properties, also of interest when

analyzing protein-protein interactions.

Krogan reported more interactants per bait than did Gavin, which could be

due to differences in handling of samples or that in addition to the MALDI-

TOF mass spectrometry method used by Gavin, Krogan also used the more

sensitive LC-MS/MS approach. By comparing with the manually curated

datasets and data on co-localization and biological process annotation, we

conclude that the quality of the Gavin dataset is slightly higher. To facilitate

comparison, we reduced the Krogan dataset to a size similar to Gavin,

whereby a similar quality was achieved.

We found that (1) abundance was the most important factor in the TAP

experiments, not surprising as abundant proteins are easier to detect with

MS. (2) Repeating domains were overrepresented among interacting proteins

in all the datasets. (3) Disordered regions were overrepresented in the Y2H

and Krogan datasets, but not in the Gavin dataset. This could be due to

differences in detection and handling of the samples between the two TAP

setups, or indicate that disordered regions not only are important for

transient interactions, but also for interactions between proteins of lower

abundance. (4) Concerning the highly interactive domains, the ten most

promiscuous ones seem to be involved in nearly half of all interactions.

We conclude that longer proteins with promiscuous domains, disordered

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54 Chapter 8 • Summary of the papers

regions and repeat domains are prone to interact frequently. However, before

deeper analysis of these properties can be carried out, the effect of protein

abundance on detection of protein interactions needs to be better understood.

Also, to ensure interaction assignments of high quality, the analysis should

be based on data stemming from a combination of different experimental

methods.

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55

8.5 Final thoughts

Not too long ago, transmembrane helices were regarded as simple anchors

made up of hydrophobic residues. Then biochemical studies indicated that it

may not be that simple. Now, with more solved three-dimensional structures,

pictures of proteins frozen in one of their conformations, we see more and

more anomalies. So many in fact, that they cannot be considered anomalies

anymore, but rather proof that we yet do not fully understand the process by

which proteins integrate into the membrane and find their functional fold.

However, the field is in steady progress.

In paper I, we searched the known structures for segments of low

hydrophobicity that still are transmembrane. We found that almost one fifth

of the helices available today are predicted not to integrate well into the

membrane, and that of the ones we investigated experimentally, most indeed

did not. This raises the interesting question as to how they are inserted.

Literature, including paper II, has some suggestions: post-translational

rearrangement, strong topological signals in loops or the previous and

subsequent helices, high concentration of polypeptides in the biological

membrane, direct interactions with neighboring helices (ion bonds, hydrogen

bonds, tight packing), etc. The last example would require the neighboring

helices to be retained in close proximity to the lateral gate, or even inside the

translocon pore. Some of the examples presented in paper I seem to be

explained by positively charged residues in the flanking loops, complying

with the strong topological “positive inside” rule. For others, the flanking

helices improved insertion (three out of five examples). For our examples

where local sequence context did not improve insertion, one TMH is known

to become integrated after a major structural rearrangement requiring the

presence of the entire protein chain, and the other one has not been reported

before. It will be interesting to follow further investigations of the exact

procedure behind the integration of these TMHs.

My belief is that the integration into the bilayer of marginally

hydrophobic helices of co-translationally integrated proteins can take many

routes and that we are now in the beginning of understanding how this

works. All the mechanisms/factors mentioned above are likely to be

involved, and it will be a challenge to decipher the “rules” for what

mechanism/factor that is decisive for each segment. The translocon

accessory proteins may also play a greater role than we presently are aware

of. With the much different mechanisms for post-translational translocation

(not integration) of proteins in eukaryotes (chaperones) and bacteria

(chaperones, SecA) in mind, it seems plausible that the mechanisms for co-

translational integration into the membrane differ at least somewhat between

different organisms, even if the protein sequence requirements seem to be

surprisingly similar. The core of the translocon, the pore, is conserved

throughout the three kingdoms of life. However, the variation in accessory

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56 Chapter 8 • Summary of the papers

proteins seems to be greater. To figure out the details of the

translocation/integration processes, more light needs to be shed on the

proteins assisting the translocon pore and how they interact with the

translocating/nascent protein chain, especially the marginally hydrophobic

segments. Post-translational rearrangements can also be expected to play a

substantial part in the integration of the latter kind of segment. The findings

that we present in paper II indicate that many segments of low

hydrophobicity could become transmembrane after the initial insertion of an

overlapping, more hydrophobic segment. It will be exciting to see if other

examples of repositioned TMHs will be presented, and also to see if the

repositioning process involves any chaperones, yet unidentified structural

signals, or just a series of spontaneous re-formations of molecular bonds due

to known factors.

One thing that I’ve learnt to appreciate during my work is the number of

databases collecting available data and presenting it in an apprehensible

form. However, when using these databases, one must always be aware of

the features of the dataset. For instance, you may have access to 100

proteins, but if all of them are homologues, you cannot really draw any

conclusions that are valid for proteins in general. For instance, we always

work with homology-reduced datasets for statistics on protein structures and

sequences. Also, what is the quality of the data, does one need to filter it

somehow? What was the method used to gain the information, is it biased

and if so, how? In paper IV we analyzed the data on protein-protein

interactions from two similarly executed large-scale investigations together

with results gained using an altogether different technique. We found that the

methods indeed were biased and that our knowledge on this was highly

useful for the interpretation of the results. We also found, as many others

have stated before, that conclusions drawn from a combination of data,

preferably stemming from orthogonal methods, are the most reliable ones.

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57

Acknowledgements

No man is an island, John Donne wrote almost four hundred years ago. We

all need others to thrive. A large number of people helped me thrive during

my years in the Arrhenius Laboratories. You know who you are and there are

many of you, but the following people deserve a special thank you:

My supervisors Arne Elofsson and Susana Cristobal, for all your ideas and

your support and dedication during these years and for the relaxed and

permissive environment you create. All these things mean a lot to me, and I

have enjoyed being a family-run venture.

Ingmarie Nilsson and Gunnar von Heijne, for your great expertise and ideas,

and for always having time to listen to problems and come with suggestions.

It has been great collaborating with you.

Erik Lindahl, for always being so enthusiastic and for sharing good advice

on both science and life in general. I always enjoy talking to you.

Anni Kauko and Yana Vereshchaga, for stimulating collaborations. Anni, you

do have an expert eye for three-dimensional structures.

Mirjam Lerch-Bader and Karl Enquist for teaching me the tricks of the trade

of rough microsomes and in vitro glycosylation. I could not have done any

of it without you.

Karin Öjemalm, for all of those clones and assays. We finally did it! It was

great working with you.

Kristoffer Illergård and Aron Hennerdal, for five years of studying, hanging

out and playing Settlers, followed by five years of PhD studies, hanging out

and publishing a paper. Who would have guessed? It will be weird working

without you. Kristoffer also for the “rolled out” structures of LacY and GltPh

in figure 14.

Åsa Björklund, for being a mentor as well as a colleague for the new girl

five years ago. We may not have a paper to show for it, but working with

you in the lab was still a rewarding experience.

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Acknowledgements 58

The lab rat gang in the crowded office. Itxaso Apraiz, for being a great

colleague and teaching me a lot about myself. Anya Amelina, for being the

sweet person that you are and for being with me since the very beginning.

Minttu Virkki, I’m so happy that Arne and Susana agreed with me that we

needed some extra hands in the lab that summer. Proteins, promenades and

parties, it has all been great fun! Maria Bendz, our very first post-doc, thank

you for having great ideas both in the lab and for whatever festivity that is

coming up and for going upstairs with me to buy coffee. Narges Bayat, the

nicest daffodil I have ever met, for teaching me that people from Iran eat

stew. Daniel Nilsson, for arguing for positive thinking and making me do it.

Gabriella Danielsson, for your awesome recipes and for all your help.

Sara Light, Niklas Barkå and Stefan Nordlund for proofreading this thesis.

Stefan also for being a rock to lean on for all us PhD Students at DBB.

Christine Sophie Schwaiger, for the pymol pictures of water and lipids used

in figures 2 and 3.

Hanna Eriksson, for all those hours discussing biochemistry in preparation

for the Big Exam. What would I have done without you?

All not previously mentioned friends, co-workers, administrators and

technical staff at the department. Thank you for making my life easier, both

in the scientific field and outside. I could not have wished for better

company at lunch breaks, cake clubs, three o’clock “fika”, late evenings at

the department, after work events, DBB pubs, conferences, dinners and so

on.

Josefin Illergård, Sandra Siljeström and Cecilia Århammar, non-DBB PhD

Students and dear friends, for lending listening ears even to work-related

babble.

My family, both original and extended. You are with me in everything I do

and I am lucky to have you in my life. Especially my parents, always

encouraging me to pursue what I want and reminding me to slow down from

time to time. Your love is my solid foundation.

Jonas, because even after having to deal with me while I wrote this thesis,

you still want to marry me. Where you are, that is home for me.

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