On the Function and Structure of Synthetically Modified Porins

Angew. Chemie. Int.Ed, 2009, 48,1-6Lyndelle LeBruin

CHEM 258

1

Outline

• Beta barrels

• Porins and ion channel engineering

• Structure and function of OmpF porin

• Main paper discussion

• Conclusions

2

βeta barrels

• Large beta sheet that twists and coils to form a closed structure: first strand is H-bonded to the last

• Beta strands arranged in an anti-parallel fashion

• Found in porins or other proteins spanning the cell membrane• Porin like barrels form 2-3% genes in Gram-negative bacteria

3

βeta barrels

• Hydrophobic residues oriented toward exterior-interact with surrounding lipids; hydrophilic residues oriented toward inner pore

4

Sucrose specific porin for Salmonella typhimurium- a canonical beta barrel protein

Beta sheet

Ion channel engineering?

• Channel mediated transport of ions through membranes has great potential in neurobiology and bio-sensing

• Transport of ions or molecules across membranes is facilitated by pores, channels.

• Ion channel engineering has two approaches: - create biological channels to control existing function: structural information of channels, chemical synthesis to

obtain hybrid ion channels - de novo design: use of self assembly to form π & β barrel pores

5

Work in progress?

• Attachment of synthetic modulators to biological ion channels

• Modifying channels with narrow ion conductance pathways

6

Progress in Ion channel engineering

7

First eg. of functional semi-synthetic K+ channelSelectivity filter for K+ ions

-Target: First 125 AA of native 160 AA protein KsCA- recombinant expression to produce 1-68 N-peptide α-thioester residues- synthetic C- peptide residues(69-125)- chemical ligation with thiophenol

Semi-syntheic K+ channel

8

- amide bond between Tyr78 & Gly79 changed to ester bond: ion selectivity unchanged, significant change in distribution of K+, Rb+, Cs+ single channel conductance reduced (50%)

- TVGYG : control flow of K+, only chemoselective method without loss

of function

- TVGYG adopts left handed helical conformation in crystal structure for D-AA, but not in L-AA

- altering of Gly77 to D-Ala, no change in channel activity

- altering of Gly77 to L-Ala: non-functional channel

Porins

• Membrane proteins• Allow diffusion of small hydrophilic molecules across

membrane of Gram negative bacteria• Matrix contains “trimers” of identical subunits with 16-

stranded anti-parallel β-barrel containing pores• Contain hollow centers through which small molecules can

diffuse• Wide at both ends, contain eyelet, central pore,extends over

~10Å in middle of membrane

Porins

Why use Porins?• Conductance pathway formed by single polypeptide chain: eases synthetic modifications in the pore interior• Pores have variable diameters, to attach synthetic modulators• Not substrate specific transporters which undergo

conformational changes for transport of molecules • Porins are unlike carriers which diffuse with their cargo

through the lipid membrane• Porins are biological ion channels: - allow flow of ions across the membrane close to diffusion limit

in water, high specificity for ion conductance

11

Structure and function of the OmpF porin

12

OmpF Porin

Secondary structure of OmpF porin monomer:

• Golden strands represent beta helix strands.

• Pink strands represent alpha helix strands.

• Gray strands are loops that connect the many beta helix strands to each other.

OmpF Porin• Origin: Escherichia coli; outer membrane protein• 340 amino acids long• 16 stranded antiparallel β barrel structure• 3 OmpF molecules assemble to trimer within pore• Tight monomer interactions increase stability of trimers• Loop region (L3) folds within pore, forms a constriction zone

(9Å) through the barrel: protect channel, screen solutes based on charge and size

• Channel of OmpF restricted to 7x11 Å diameter: lined with basic and acidic residues

• Pore allows passage of molecules of up to 600Da, minor ion specificity

14

Pore constriction of wild type OmpF

15

Stucture of OmpF

a: side view of OmpF momomerb: top view of the OmpF trimer with loop region L3β1: peptide stretch for native chemical ligationLys16: site for attachment of synthetic modulators

16

The Design of the modulator

• N terminal region covering β strands 1 and 16 selected for synthetic modification

• Side chain of Lys 16 faces constriction zone, used for attachment of modulators

• Modulators attached covalently via : - protein semi-synthesis via NCL/click chemistry - cysteine residue formed via mutation and then S-alkylation

17

Native chemical ligation(NCL)

• Construct large polypeptides from two or more unprotected peptides• Peptides w/ C terminal thioester reacts with peptide containing an N

terminal cysteine in presence of thiol catalyst• Performed in aqueous solution

18

Synthesis of OmpF Derivatives

OmpF Hybrid 5 19

- Synthesis by NCL 1: Lys16 changed to propargyltyrosine ether П(K16П)

- C terminal OmpF fragment 4 formed as inclusion bodies with porin deficient E.Coli strain

- NCL of 3 and 4 under denaturing conditions (8M urea) - Ligation yields (50%) comparable to native analogue of 4 with Lys16 (60%)

Synthesis of OmpF Derivatives

20OmpF hybrids 8 & 10

- OmpF mutant Lys16 modified to cysteine (K16C)

- S-alkylation of 6 with 7 gives 8 in good yields(90%)

Preparation of recombinant OmpF and OmpF fragments

21

Refolding of OmpF hybrids

• Hybrids refolded by insertion into mixed unilamellar vesicles with a 1:1 ratio of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and n-dodecyl-β-D-maltoside using the procedures for the unmodified OmpF protein

• Bulky dansyl groups in pore gave refolding yields similar to unmodified OmpF (70%)

• Hybrids 5 & 8 further purified from unfolded protein by trypsin digestion, shows stability of intact OmpF trimers against proteolytic digestion

22

SDS resisitant membrane OmpF

23

Trimers remain stable after tryptic digestion

Preparation of functionalized OmpF porins by NCL

24

- Refolding yield after ligation and purification(~70%) comparable to unmodified OmpF- Hybrids 5 & 8 can be re-purified from unfolded protein by trypsin digestion, similar to unmodified OmpF – no proteolytic digestion

Preparation of OmpF porins by S-alkylation

Refolding yield (~70%), comparable to that of native OmpF

Porin modification

• Functional consequences determined by conductance measurements

• High salt concentrations used to study blockage efficiencies versus differences in ion channel electrostatics

• Black lipid membrane (BLM) technique at +140mV and I/U curves used to track single pore closure within OmpF trimer

26

Porin modification

Hybrid 5: Not a significant change in I/U values for recombinant OmpF,

wild type OmpF by NCL and OmpF hybrid 5 51.5 pA vs. 51.2 pA for OmpF

Modulator(319Da) near constriction zone does not alter conductance of OmpF, good method for constructing native-like OmpF polypeptides

Inspection of trimer events forOmpF derivatives

28

Cysteine linked dansyl OmpF hybrid 8

29

BLM measurements in 5 mM HEPES, pH 7.2; 150 mM KCl

Large spread of trimer conductances, seen for hybrid 8,were not observed for refolded wild type OmpF or its mutants

Average specific conductance of 8 decreased by 15% 0.78 nS vs. unmodified OmpF 0.92 nS

Indicates conformational mobility and heterogenity of modulator within the pore

OmpF hybrid 10

Size of hybrid 5 vs.hybrid 8 (451 vs. 377 Da) not sufficient to determine effect of modulator on channel

I/U for 10 similar to that of hybrid 8: large spread in trimer conductances, 18% (0.75 nS) conductance reduction vs. 15% (0.78 nS) for hybrid 8, limiting conductances of 0.42 and 1.14 nS Single trimer events, show reduced conductivity for hybrid 10

30

Crystal structure of OmpF Hybrid 10

31

Top view of OmpF trimer with dibenzo-[18]crown-6 ether modulator

Side view of the cross section of 10

X ray crystal structure of hybrid 10

• Two trigonal crystal forms obtained, one molecule per asymmetric unit

• Crystal structure I, diffraction 3.2 Å similar to known crystal form of OmpF, lacked ordered density for synthetic modulator

• Crystal structure II diffracted to 3.4 Å, first report to date of pore blocked by partially by modulator

• OmpF trimers form columnar structures along the c axis

32

Crystal structure of OmpF Hybrid 10

33

Fobs - Fcaldifference density map at 3.4 Å Crystal packing of novel OmpF crystal: quasi-continuous arrangement of trimers along “c” axis

Surface representation for dibenzo[18] crown 6 compound between L3 loop and basic amino acids

Crystal structure II

• Protein conformations of structures are identical• Crystal structure II: difference in electron density in ion conductance

pathway of OmpF channel• Explanation for electron density difference: - ether crown moiety from Cys16 transverses constriction zone - distorted conformation of ether moiety in constriction zone• Sulfonate group on HEPES interacts with basic patch of pore interior• OmpF wide pore channel has been altered by synthetic modulator,

occupied a closed state, trimers have divergent conductance properties

34

Blockage of the constriction zone with OmpF hybrid 10

35

Blocked (orange) and loosened (light yellow) conformations

Stretched, inward-oriented conformation of crown-ether required for blockage: partial contact of L3 loop

Crown ether points away form constriction zone : more conformational freedom, no pore blockage

Linker between crown ether and position 16 on β-strand 1 is extremely important in determining efficiency of channel or pore blockage

What do the results show?• Conformational heterogenity of hybrid channels no longer a

sole consequence of properties of protein template

• Protein template and synthetic modulator interplay necessary for maintaining conformation within the pore

• Single site attachment of modulators in large pores does not solely change properties(electrophysiological) of pore

• For effective pore blockage, additional non-covalent interactions and second site attachment of modulator to pore is necessary

36

Conclusions

• Ion channel engineering

• Porin structure and function

• OmpF porin as an ion channel modulator

• Future directions

37

Acknowledgements

• Dr. Martin Case

• Chem 258 class

• Peace of mind

38

Black lipid membrane (BLM)

Electrical characterization has been particularly important in the study of voltage gated ion channels which can be inserted into a BLM by coating them with a detergent and mixing them into the solution surrounding the BLM.

The term “black” bilayer refers to the fact that they are dark in reflected light because the thickness of the membrane is only a few nanometers, so light reflecting off the back face destructively interferes with light reflecting off the front face.

To make a BLM, a small aperture is created in a hydrophobic material such as teflon. A solution of lipids dissolved in an organic solvent is then applied with a brush or a syringe across the aperture