24 January 2005

Biol 6312

Introduction to the course and the topics

Interplay of protein structure <=> function and their relationship to design

Does a particular structure lead to a particular function?
Does a particular function require a particular structure?

Design:
Can we design a protein with a given structure?
Can we design a protein with a given function?

	Written on the blackboard in his office at Caltech
Richard Feynman:	"What I cannot create, I do not understand."

What is a protein?

A linear polymer of amino acids
A globular protein has a defined, compact shape: a conformation

But it is not merely collapsed. It is very densely packed: an aperiodic crystal
The density of a crystal, without a repeating pattern.

Of course, some proteins are fibrous or unstructured, or they contain elements that are fibrous or unstructured. We will focus on the globular ones.

What are the principles of protein structure?

Jane Richardson has said that proteins are interesting because they are inherently one- and three- dimensional. (Richardson Lab)

The attempt to understand this relationship is know as:

The protein folding problem

What is the language of protein folding?

DNA sequence --> Protein sequence (Genetic code)

Protein sequence --> Folded protein (Protein folding code)

The human genome has now been sequenced. Also many other organisms.

Will we need to crystallize all 25,000 or 50,000 human proteins?

Or, will we be able to apply knowledge of the principles of protein structure, and knowledge of similar proteins, to make reasonable predictions of structure?

Two interesting observations with respect to the language of protein folding:

1. Random sequences of amino acids usually will not adopt a compact, folded structure. Sometimes they will adopt a loosely folded structure.

"Random words seldom make a sensible sentence."

2. Most proteins that have a compact, folded structure have an amino acid sequence that is "over-determined". That means many amino acid replacements (mutations) will not significantly disrupt the 3-D structure.

"A single misspelled or mispronounced word may not obscure the meaning of a sentence."

Why are proteins so robust to site mutations?
J Mol Biol 2002 Jan 18;315(3):479-84
Taverna DM, Goldstein RA.

What are the properties of the 20 amino acids that are important to protein folding?

1. Polarity (charge, H-bonding capacity, hydrophobicity)
2. Stereochemistry (size and shape)

1) Polarity
Classical review of Walter Kauzman Advances in Protein Chemistry (1959) 14:1-63.
Some factors in the interpretation of protein denaturation.

Polar side chain amino acids are on the surfaces of proteins
Hydrophobic side chains are in the interiors

Burying of the hydrophobic side chains provides the largest stabilizing term in the ∆G of folding. This is due to the increase in entropy of the water, as it is released from its interactions with the hydrophobic surfaces. Figure 1-34.

Termed the"hydrophobic effect" by Charles Tanford

Tanford C.
How protein chemists learned about the hydrophobic factor.
Protein Sci. 1997 Jun;6(6):1358-66.

How to rank the amino acids in terms of polarity, or hydrophobicity?

1. Calculation of hydrophobic surface
2. Measure ∆G_transfer from water to organic solvent
3. Statistical analysis of locations of each aa type in a structural database

Example

2) Size and Shape has great influence on protein conformation

Note that many side chains are similar or identical to others in size or shape.

Conformational flexibility of the peptide chain

Proteins are made from peptide bonds

between amino acids

There are 3 torsional angles for each amino acid in a polypeptide.
N-C φ C-C Ψ , C-N ω

1. Peptide bond, ω (References below)

Considered to be 180˚ because of double bond character. Two types of deviation:

A. Near 180˚: mean of 187 proteins, 174˚ ± 4.5˚ SD, min 117˚, max 260˚

Deviations from Planarity of the Peptide Bond in Peptides and Proteins
Malcolm W. MacArthur, Janet M. Thornton
J. Mol. Biol. (1996) 264 pp. 1180-1195

B. 0˚ or cis rather than the more common trans

Only about 1-2 % of peptide bonds in proteins are cis. That's because of the unfavorable free energy of bringing the backbone close together (X-Xnp). Unless the second aa is Proline. Then the difference between cis and trans is not so great, so trans is not as highly favored. Consecutive small amino acids sometimes make a cis peptide bond. With Pro cis bonds, the preceding aa is often aromatic.

Cis Peptide Bonds in Proteins: Residues Involved, their Conformations, Interactions and Locations
Journal of Molecular Biology, Volume 294, Issue 1, 19 November 1999, Pages 271-288
Debnath Pal and Pinak Chakrabarti

The partial charge of the nitrogen atom in peptide bonds
EJ Milner-White
Protein Sci (1997) 6: 2477-2482.

The nitrogen is not actually positive, but this is only a formal charge. The calculated charges are: H (+ .2 to + .4), N (- .2 to -1.2), O (- .4 to -1.1), C (+ .4 to + 1.4) This makes the peptide bond dipolar, and the α helix also dipolar.

2. Φψ angles

These torsion angles are often displayed on a Ramachandran plot. An entire protein's values, or many proteins, can be diplayed on one diagram.

RAMACHANDRAN GN, RAMAKRISHNAN C, SASISEKHARAN V. Stereochemistry of polypeptide chain configurations. J Mol Biol. 1963 Jul;7:95-9. G.N Ramachandran died in April 2001 (story)

There are 3 discrete regions of allowed torsion angles due to steric conflicts between C=O groups and side chains. Allowed regions correspond to the well-known secondary structures.

Disllowed regions, as studied below, are generally populated by Glycines

Disallowed Ramachandran Conformations of Amino Acid Residues in Protein Structures
K. Gunasekaran, C. Ramakrishnan, P. Balaram
J. Mol. Biol. (1996) 264 pp. 191-198

3. Secondary structure: patterns of Φψ values

Can be repeating (α-helix, β-sheet, 3₁₀ helix, polyproline helix, left-handed α-helix) Historical account by David Eisenberg (PubMed)

or non-repeating (turns, loops)

Can be defined in terms of torsion angles of each residue
repeating (same), nonrepeating (each residue may be different)

Strength of interactions in proteins: Figure 1-10

4. Hydrogen bonding

Patterns of hydrogen bonding also are an indication of secondary structure.

H-bonding is important kinetically in the protein folding process

In a stably-folded protein we expect that all possible H-bonds will be made.
It appears to be 90-98% are H-bonded.

McDonald IK, Thornton JM.
Satisfying hydrogen bonding potential in proteins.
J Mol Biol. 1994 May 20;238(5):777-93.

H-bond partners include backbone groups (>NH and >C=O) and also polar side chains. They can H-bond to each other or to the solvent (H₂O).

Backbone groups are typically H-bonded by the repeating patterns of secondary structure.

Side chains are typically H-bonded to solvent or to each other.

Strength of H-bonds depends upon both angle and distance

>N-H---O=C< Here, NH is the donor and O is the acceptor

N-O distances are typically .28 to .31 nm (2.8Å to 3.1 Å)

A shorter H-bond is stronger because it is an electrostatic interaction.

The angle can be important, depending upon the hybridization of orbitals

For 2 donors, 120˚ bonds are favored, while for one donor, 180˚ bonds are favored. NH is always preferred in the plane of CO.

From the following paper a summary of actual H-bonding in a large collection of proteins can be examined.

Hydrogen bonding in globular proteins (Medline)
Stickle DF, Presta LG, Dill KA, Rose GD
J. Mol. Biol (1992) 226:1143-59

1) Most H-bonds are local---between residues that are close in sequence. (Exception: ion pairs)

2) Most H-bonds are between backbone groups (68%)

3) 82% of the amino acids in H-bonds were in regular secondary structures.

4) Breakdown of backbone H-bonds:

37% i -> i+3 (helices, turns)
32% i -> i+4 (helices)
26% more distant (β sheets)

5) Most side chain to backbone H-bonds are local, e.g. helix capping

6) Network H-bonds are common More than one donor and/or acceptor (stronger)

C-H...π-Interactions in Protein
Journal of Molecular Biology, 307: 357-377
Maria Brandl, Manfred S. Weiss, Andreas Jabs, Jürgen Sühnel and Rolf Hilgenfeld

Strength of the Calpha H..O hydrogen bond of amino acid residues.
J Biol Chem 2001 Mar 30;276(13):9832-7 Scheiner S, Kar T, Gu Y.

Recent studies have indicated that C-H groups can be H-bond donors, although they are considered to be weak. S-H groups can also be weak donors or aceptors. O-H groups are strong donors or acceptors. N-H groups are strong donors, but only some N's (without H) are acceptors.

Aromatic groups can also be acceptors. In the study below, 1 aromatic residue in 10.8 was found to be in such an interaction (about 2 per protein)

O-H, N-H, S-H, and C-H to Trp, Tyr, Phe, His

to Trp was most common ( 1 in 5.7)

They seem to be common at the ends of helices and strands.

They are considered secondary acceptors, not as good as the others, bonding is not as strong.

Hydrogen Bonds with π-Acceptors in Proteins: Frequencies and Role in Stabilizing Local 3D Structures
Thomas Steiner, Gertraud Koellner
J. Mol. Biol (2001) 305: pp. 535-557

5. Ionic Interactions

Cation-interactions with aromatic groups are stronger than just H-bonding.
These typically involve Arg or Lys with Trp (most common), Tyr, or Phe.

Cation-π interactions in structural biology
Justin P. Gallivan and Dennis A. Dougherty
P.N.A.S. (1999) 96: 9459-9464

Standard ion pairs are between positively-charged groups Arg, Lys, or His and negatively-charged ones, Asp or Glu. They can make strong interactions, but in the context of a protein, they stabilize the folded state to various degrees, depending upon details.

Ion pairs are often called salt bridges. They can be simple (2 partners) or complex (network, several partners, linear or branched)

Complex Salt Bridges in Proteins: Statistical Analysis of Structure and Function
Boaz Musafia, Virginia Buchner, Dorit Arad
J. Mol. Biol. (1995) 254: pp. 761-770

According to the analysis of Musafia, in 94 analyzed proteins

1) 1/3 of all residues in salt bridges are in complex ones or 3 or more.

2) the same geometry is used in complex salt bridges, the extra residues are just added.

3) A common rule is that they are found between twwo subunits, or are between 2 different elements of secondary structure. An exception is that they form along an α-helix, residue i-> i + 3 or 4

Secondary Structure

1. 1. α-helix (Figure 1-13)

1. H-bonding pattern: CO (i) to NH (i+4)

1. 3.6 residues per turn

1. φ = - 57˚ ψ = - 47˚

   Side chains protrude out from helix axis (Figure 1-15).

   Amides are oriented to make a dipole.

   Jmol α-helix

1. Other helices (Figure 1-14):

   3₁₀ helix (rare) 3 residues per turn. More tightly wound than α-helix

   H-bonding pattern: CO (i) to NH (i+3)

   π helix (extremely rare)

2. Polyproline II helix (like collagen)

3. Left-handed Polyproline II Helices Commonly Occur in Globular Proteins

4. Alexei A. Adzhubei, Michael J.E. Sternberg

5. J. Mol. Biol. (1993) 229: pp. 472-493

6. 2. β-sheet (Figure 1-17)

   A -βsheet is formed from 2 or more β-strands.

   The strands are nearly extended (-180, +180), but the actual angles cover a rather wide range of values centered at approximately (-130, +125)

   Backbone H-bonding occurs between adjacent strands

   Sheets can be parallel, anti-parallel, or mixed.

   The strands tend to be twisted in a right -handed sense due to the L-form of the amino acids.

   The side chains protrude away from the sheet, in an alternating way: 1-up, 2-down, 3-up, 4-down etc. β-sheets can be amphipathic also.

   β-sheets are sometimes imperfect. For example one residue might not H-bond to another strand according to the rules. This forms a β-bulge.

   
   Chan AW, Hutchinson EG, Harris D, Thornton JM.
   Identification, classification, and analysis of beta-bulges in proteins.
   Protein Sci. 1993 Oct;2(10):1574-90.

   3. Turns, loops, connections

1. These connect elements of repetitive secondary structure.

   Turns are the shortest, consisting of 3-4 amino acids usually. Sometimes called β-turns. They have been classified in the following paper into about 7 groups: I, II, VIII, I', II', VI, IV

   Hutchinson EG, Thornton JM.
   A revised set of potentials for beta-turn formation in proteins.
   Protein Sci. 1994 Dec;3(12):2207-16.
   

Omega loops connect 2 elements of secondary structure, but follow a path that is longer than necessary, shaped like the Greek letter Ω.

Fetrow JS.
Omega loops: nonregular secondary structures significant in protein function and stability.
FASEB J. 1995 Jun;9(9):708-17. Review.

Pal M, Dasgupta S.
The nature of the turn in omega loops of proteins.
Proteins. 2003 Jun 1;51(4):591-606.

Connections are typically longer segments or irregular structure that connect 2 distant elements of secondary structure.

How long are α-helices and β-strands on average? See handout for the distributions.