Basics of protein structure and stability IV: Anatomy of protein

structure continued

Biochem 565, Fall 2008

09/03/08

Cordes

the main-chain can hydrogen bond to itself

the amide nitrogen:

main-chain hydrogen bond donor

the carbonyl oxygen:

main-chain hydrogen bond acceptor

there are also side-chain acceptors and donors

N

CH

C

CH3

O

N

CH

C

H2C

O

O

H

HH

• Hydrogen bond not really a covalent ”bond”--not much orbital overlap.

• Model as an electrostatic interaction between two dipoles consisting of the H-N bond and the O sp2 lone pair. In electrostatic theory, the optimal orientation of two such dipoles is head-to-tail. The energy of such an arrangement should decrease as the head and tail are brought together as long as atomic van der Waals radii are not violated (then repulsive forces quickly take over).

• “Ideal” hydrogen bond in this model would have r~3.0 Å, p=180°, =0° and =±60°. Convince yourself of this.

• In small molecule crystals, this is approximately what is observed, though there is a lot of variation in the angles and . Thus the precise C=O…H angle parameters are not critical.

• Main chain-main chain hydrogen bonds found in proteins will show various deviations from this geometry, partly due to the topological constraints imposed by forming secondary structures.

Hydrogen bond

geometry

• What is a “reasonable” hydrogen bond? Criteria for identifying hydrogen bonds are somewhat arbitrary and many have been used. Here are a couple of examples.

• Geometric criteria: Often H-bonds are just identified by two parameters, the O…N (acceptor-donor) distance r, and a O…H-N angle p. The angles describing the C=O…H geometry are sometimes ignored. Typical cutoffs: p > 120° and r < 3.5 Å. (Baker & Hubbard, 1984)

• Electrostatic criteria: One of the most commonly used criteria is a potential function based on a pure electrostatic model (Kabsch & Sander, 1983). Place partial positive and negative charges on the C,O (+q1,-q1) and N,H (+q2,-q2) atoms and compute a binding energy as the sum of repulsive and attractive interactions between these four atoms:

E=q1q2(1/r(ON)+1/r(CH)-1/r(OH)-1/r(CN))*f

where q1=0.42e and q2=0.20e, f is a dimensional factor (=332) to convert E to kcal/mol, and r(AB) is the interatomic distance between atoms A and B.

A hydrogen bond is then identified by a binding energy less than some arbitrary cutoff, e.g. E< -0.5 kcal/mol.

• Note that the criteria defined above are only applicable when hydrogen atom positions are available. Crystal structures do not have hydrogens--however, their positions can be computed in many cases.

Identifying main-chain H-bonds in X-ray structures of proteins

AAON

DD

D'D ≤ 3.5 Å

90-150° 110-180 °

DD, DD' = donor antecedent a tomsAA = acceptor antecedent a toms

0-20°

tors ion angleN-DD-DD'-Odescribes degreeto which acceptor oxygenout of plane with the nitrogen donor

Presta LG & Rose GD Science 240, 1632 (1988)

X-ray structures of proteins do not in general include hydrogen atom coordinates--get used looking at pictures of proteins without the hydrogens, and having your mind fill them in

Secondary structure elements in proteins

beta-strand(nonlocal interactions)

alpha-helix (local interactions)

A secondary structure element is a contiguous region of a protein sequence characterized by a repeating pattern of main-chain hydrogen bonds and backbone phi/psi angles

reflect the tendency of backbone to hydrogen bond with itself in a semi-ordered fashion when compacted

Local backbone H-bonding: DSSP turn/helix definitions3-turn: ‘>’ ‘3’ ‘3’ ‘<‘ notation-N-C-C--N-C-C--N-C-C--N-C-C- residues H O N O H O H O >----------------< H-bond

4-turn: ‘>’ ‘4’ ‘4’ ‘4‘ ‘<‘ notation-N-C-C--N-C-C--N-C-C--N-C-C-N-C-C residues H O N O H O H O H O >----------------------< H-bond

5-turn (just an elaboration of 3- and 4-turn.

A minimal helix is two consecutive N-turns--for a minimal four helix from residue i to i+3: i <--residue>444< and >444< overlap to give>4444< which defines a helix HHHH from i to i+3 ‘H’ is the notation for a residue in a 4-helix.Notice that the helix does not include the residues involved

in the terminal H-bonds.

Longer helices are overlapping minimal helices.

Kabsch & Sander, 1983

the alpha-helix: repeating i,i+4 h-bonds

2

1

3

4

5

7

8

9

6

10

11

12

By DSSP definitions, which of residues 1-12 are in the helix? Does this coincide with the residues in the helical region of phi-psi space?

right-handed helical region of phi-psi space

hydrogen bond

The -helix, with i,i+4 h-bonds, is not the only way to have local hydrogen bonding of the backbone to itself.

The 310 helix has hydrogen bonds between residues i and i+3

The helix has hydrogen bonds between residues i and i+5.

For a number of reasons almost all helices in proteins are -helices--include backbone, side chain steric issues, van der Waals contacts, H-bondgeometry

-helix 310 helix helix

these are poly-Ala,so the gray balls on theoutside are -carbons from the side chains

310 and helices have sterically allowed conformations but not in the most favored

regions of phi-psi space

Helix nomenclature: -helix example

1 Hydrogen bond betweenC=O H-N

(residue i) (residue i+4)

15 helix

2 Repeating unit: – 5 turns

– 18 residues per repeat

185 helix3 Loop formed between C=O H-N

– 13 atoms 1 5

– 3.6 residues per turn

3.613 helix helices extend with approximately 1.5 Angstrom per residue, 5.4 Angstrom per turn.

nomenclature used for 310 helix

Nonlocal backbone hydrogen bonding:

DSSP bridge, ladder and sheet definitions

parallel bridge:

‘x’ notation

-N-C-C--N-C-C--N-C-C- residues H O H O H O \ . . / H-bonds \. ./ (\ and /, .\ /. or .) . \ / . H O H O H H residues-N-C-C--N-C-C--N-C-C- ‘x’ notations

antiparallel bridge:

‘X’ notation

-N-C-C--N-C-C--N-C-C- residues H O H O H O . ! ! . H-bonds . ! ! . (! or .) . ! ! . . ! ! . O H O H O H residues-C-C-N--C-C-N--C-C-N- ‘X’ notations

ladder= set of one or more consecutive bridges of identical typesheet= set of one or more ladders connected by shared residues

Kabsch & Sander, 1983

beta strands/sheets

Is this a parallel or anti-parallel sheet?

49

50

51

52

53

54

57

56

beta-strand region of phi-psi space

By DSSP definitions, which of res 49-57 are in the sheet? Does this coincide with the residues in the beta-strand region of phi-psi space?

Principal types of secondary structure found in proteins

Repeating () values

-63o -42o

-57o -30o

-119o +113o

-139o +135o

-helix(15) (right-handed)

helix(14)

Parallel -sheet

Antiparallel -sheet

All the most commonsecondary structureconformations fit nicely within sterically allowed regions of phi-psi space

parallel beta-sheetantiparallel beta-sheet

alpha-helix

Nelson et al (Eisenberg lab), Nature 435:773 (2005).for background on “polar zippers”: Perutz et al. PNAS 91:5355 (1991)These types of fibrils important in Huntington’s disease etc

amyloid-like fibril(left) of peptide GNNQNNY from the yeast prion protein Sup35, and itsatomic structure (right)

Because of the repetitive nature of secondary structures, and particularly beta-sheets, proteins can form fibrillar structures and aggregates

amide stacks

fibril axis

in the case of this fibril the side chains also hydrogen bond to each other

Fibrillar helical structures: the leucine zipper

GCN4 “leucine zipper” (green) bound asa dimer (two copies of the polypeptide) to target DNA

The GCN4 dimer is formed throughhydrophobic interactions betweenleucines (red) in the two polypeptide chains

Leu

Leu

Are main-chain H-bonds why proteins are special?

“It would seem extraordinary that no other polymer structures exist in which internal hydrogen bonding can give rise to periodically ordered conformations, but no others have been found thus far. We are therefore forced to recognize the uniqueness of this capacity in polypeptide chains, one which enables them to meet the exacting and sophisticated demands of structure and function”

--Doty P, Gratzer WB in Polyamino acids, polypeptides and proteins, pp. 111-118, 1962, University of Wisconsin Press

see also Honig F & Cohen FE Folding & Design 1, R17-20 (1996).

Globular proteins

• Keep in mind, however, that if that were all proteins could do, they would just form regular repeating structures. Instead many proteins have globular structures consisting of short secondary structure elements connected by loops and turns that are not necessarily characterized by repeating hydrogen bond structures, but which serve in part to reverse the direction of the polypeptide chain.

loops and turns

-turns in proteins:reversing the chain direction

-turn consists of two residues, where there is a hydrogen bond between the carbonyl of the residue preceding the turn and the amide nitrogen following the turn. There are a number of ways to configure the backbone to achieve this.

turn residues

direction ofpolypeptide chain

four basic tight -turns that all yield an i,i+3 hydrogen bond[from Wilmot CM & Thornton JM J Mol Biol 203, 221 (1988)]

-turn type

name

pos i+1

pos i+1

pos i+2

pos i+2

I -60 -30 -90 0

I’ +60 +30 +90 0

II -60 +120 +80 0

II’ +60 -120 -80 0

Side chainconformation

• side chains differ in their number of degreesof conformational freedom(some don’t have any, such as Ala and Gly)

•but side chains of very different size can havethe same number of angles.

Side chain conformations--canonical staggered forms

t=trans, g=gauchename of conformation

IUPAC nomenclature:http://www.chem.qmw.ac.uk/iupac/misc/biop.html

glutamate

Side chain angles are defined moving outward from the backbone, startingwith the N atom: so the 1 angle is N–C–C–C, the 2 angle is C–C–C–C

H

H CCO

HNH

C HCO

HN

C

H HCO

HN

NHCHC

CH2

O

CH2

C

O

O

1

χ2

χ3

β

γ

α

δ

χ1 = 180° χ1 = +60° χ1 = -60°

t g+ g–

Newman projections for 1 of glutamate:

Rotamers

• a particular combination of side chain torsional angles 1, 2, etc. for a particular residue is known as a rotamer.

• for example, for leucine, if one considers only the canonical staggered forms, there are nine (32) possible rotamers: g+g-, g+g+, g-g-, g-g+, tg+, g+t, tg-, g-t, tt

• not all rotamers are equally likely. • for example, valine prefers

its t rotamer (picture at right) distribution ofvaline rotamersin protein structures(from Ponder & Richards, 1987)

1801=0 360

C2

H C

CO

HN

1=180°, trans or t

Side chain rotamers are not limited to canonical eclipsed

forms--there are many subtly different rotamers

How many rotamers there are also depends on how you define whether two conformations represent different rotamers:An “x degree rotamer” in this figure means that at least one side chain anglediffers by x degrees: hence classifying rotamers by a 10 degree difference standard is finer grained than classifying them by, say, a 40 degree standard

from Xiang & Honig, 2001

This figure simply shows that the more structures you examine, the more different rotamers become apparent--so as databases of structure have increased, so has the richness of our understanding of side chain conformation.