3 Å

i i+1 i+2

Cα

CαCα

The (extended) conformation

• General shape

C’

top view

side view

N’

The (extended) conformation

• Hydrogen bonds

The β conformation

Some facts and statistics:

1. ~30% of globular proteins

2. Strands lie side by side to form a sheet

3. Up to about10 residues per strand

4. 3Ǻ rise per residue

5. Sidechains project upwards and downwards

6. Main-chain amides and carbonyls of different strands H-bond to reduce polarity

7. The sheet has a ~30º right twist

8. The sheet can be parallel, anti-parallel (β-meander) or mixed (only 20% of sheets)

9. Backbone amide-carbonyl H-bonds occur between strands

Main-chain representation

(β-meander)

The (extended) conformation

• Structural motifs

β-α-β motif Twisted β-sheet (thioredoxin)

The (extended) conformation

• Structural motifs

β-barrel

Why are helices and sheets so common?

The energy change (in kcal/mol) of transferring a charged sphere from water into a hydrophobic environment:

ΔE = El – Ew = 166 (q2/r) (1/εl - 1/εw)

c

+

Why are helices and sheets so common?

Why build helices and sheets?

• By pairing polar main-chain amide and

carbonyl groups in H-bonds, helices/sheets

electrostatically mask them from the

hydrophobic core of the protein

Disruption of one H-bond in the protein core:

+5.3 kcal/mol (Ben-Tal et al, 1996)

Disruption of 20 H-bonds (average helix):

+106 kcal/mol

Since the proteins are only marginally stable (5-20 kcal/mol), this

means the disruption of protein structure!

1 2

3

4

Reverse turns and loops

β-turns locations

β-turn structure

Loops

Antigen binding site including 6 loops

Loops• Connect secondary structure elements that create the hydrophobic core of the

protein

• Usually hydrophilic and face the outside of the protein

• Hydrophilic nature results from polar residues and fewer satisfied main-chain H-

bonds

• Often create binding/active sites of receptors and enzymes

Secondary elements are more ordered than loops

Tertiary Structure

FibrousGlobular

Two types of proteins

Unfolded Folded

Folded globular proteins have nonpolar core and overall polar surface

• Globular proteins play different roles in numerous and diverse

cellular activities (enzymes, transporters, immune, and regulatory

proteins)

• This requires some properties that can only be conferred by the

globular shape

Globular proteins

The globular shape allows secondary structures to go in different directions

This allows the protein to achieve:

1. Compactness (an advantage in the extremely dense cytoplasm)

2. Keeping hydrophilic residues outside (confers water solubility) while

maximizing the burial of hydrophobic parts

3. Easy for creating binding sites (cavities)

4. Allows the joining of functional residues that are separated by sequence

Creation of binding site from residues separated by sequence

Some basic characteristics of tertiary structureInteractions that stabilize 3D structure:

1. Covalent interactions (disulfide) – less frequent because they limit protein dynamics

2. Non-covalent – vdW, electrostatic (ionic, H-bond), non-polar (hydrophobic)

[reversible, confer specificity and allow dynamics]

Aromatic ring stacking

The Ca2+-binding ‘EF-hand’ motif (Lewit-Bentely and Rety (2000))

• Ca2+ is involved in many signaling pathways in the cell, as well as in

muscle contraction

• Ca2+ works by binding to signaling proteins (e.g. calmodulin) and inducing

conformational changes that allow further binding to other signaling

proteins

Helical motifs: helix-turn-helix

a

bX, Y: Asp/GlnZ: Asp/Gln/SerY: Main-chain carbonylX: H2OZ: Asp/Glu

Helical motifs: helix-turn-helix

• EF-hand (Ca2+ binding)

• The Ca2+ binding motifs in proteins are of

limited configurations

• The most common motif is the ‘EF hand’,

adopted also by bacteria

• This motif was first discovered in the

muscle protein Parvalbumin (Krestinger) (Lewit-Bentley and Rety 2000)

1.1 Helix-loop-helix (HLH) motifs

• The motif is formed by the 5th and 6th helices (termed E, F) in

parvalbumin, hence the name

• Based on this structure and the sequence constraints emerging from

it, Krestinger predicted EF hand motifs in troponin C and

calmodulin, which were later confirmed

(Lewit-Bentley and Rety 2000)

• The ‘hand’ analogy describes both the fold (helix-loop-helix) and the motion

induced by Ca2+ binding (a)

• The Ca2+-binding loop usually includes 12 residues with the pattern X•Y•ZG–Y•–

X••–Z, where X, Y, Z, –X, –Y and –Z are the ligands that participate in metal

coordination (b) and “•” marks any amino acid

• In Parvalbumin, Ca2+is coordinated by the carboxylate sidechains of 5 residues

(Asp/Glu), by main-chain carbonyl groups and by H2O

• The 6th residue of the loop is Gly, preventing disturbance to the structure

X, Y: ~ D/NZ: ~ D/N/S-Y:~peptide carbonyl-X:~ water-Z:~E/D

(Lewit-Bentley and Rety 2000)

• Some EF-hand motifs cannot bind Ca2+ (e.g. p11). In these, the EF-

hand conformation is maintained in the ‘open’ (analogous to Ca2+-

loaded) form by a network of H-bonds (c)

• The motif is detected in small proteins (e.g. calmodulin), or within

the domains of larger proteins (e.g. myosin or calpain)

• EF-hand motifs usually occur in pairs (two, or four in a dimer), with

cooperative binding

cd

Ca2+

Free Bound

myosin light chain

Helical motifs: helix-turn-helix

• The EF-hand motif in calmodulin (CaM)

e f

Helical motifs: helix-turn-helix

• The EF-hand motif in CaM

• DNA-binding proteins (e.g. transcription factors) are able to

recognize nucleotide sequences both specifically and non-

specifically (the difference results from affinity)

Helical motifs: helix-turn-helix

DNA

TF

Helical motifs: helix-turn-helix

• Helix-turn-helix (DNA binding)

• Direct read-out of the DNA usually

requires the protein to penetrate into

the major and/or the minor grooves of

the DNA

• One way to achieve this type of

penetration is by using HTH motifs

(the connection here is a short β-turn)

• The β-turn and first helix position the

second helix in an orientation that

allows it to fit inside the major groove

of the DNA

DNA-binding HTH motifs

• DNA-binding HTH proteins are used

by both bacteria and eukaryotes

• In eukaryotes, they serve in

developmental regulation of gene

expression

• Such are the ‘homeodomain proteins’,

which contain an extended HTH motif

DNA-binding HTH motifs

3 2 1 4

motifs

β hairpin

β meander

Greek key

β-sheetβ-sheet

motifs

• The sandwich motif

VL

CL

VHCH1CH2

CH3

heavy chains

light chains

antigen binding site

• The immunoglobulin motif

motifs

Other motifs

propeller helix