3-D Structure of Proteins

Our knowledge/understanding of proteinstructure is somewhat mature. This haspermitted development of drugs based on 3-Dstructure of target proteins. (For example seethe HIV protease-nelfinavir complex: pdb code1ohr)

1

Six main protein structure themes:

1. 3D structures are determined by aa sequence2. Function depends on structure3. Most proteins exist in one (or a few) structural

forms4. The most important forces determining

structure are non-covalent (weak interactions)5. Even though there are many different proteins,

most of them have structures that fit arecognizable pattern

6. Protein structures are not completely static

2

I. Overview of Protein Structure

A. Protein conformation maintained by weakinteractions. (Conformation: eclipsed & staggered)

1. A proteinconformation of atleast moderatestability can beviewed as aminimum in anenergy well.

Textbook is fancier (Fig 4-29) 0

2

4

6

8

10

12

14

16

0 5 10 15 20 25 30 35

?G°

range of conformations

Energy conformation relationships

3

2. What is ΔGE equal to?

ΔGE = ΔHE ! T ΔSE Visualize (verbalize?) What each of these means in the

context of protein conformation:ΔGE ΔHE Ionic bonds, dielectric const?ΔSE In general terms?

Don’t forget the Δ, i.e., there are 2 states to compare.

Note: Many ideas, few pictures on pp. 116-117. Take good notesand think about the ideas thoroughly!

4

This is a turkeypancreatic peptidehormone. (pdbcode: 1ppt).

It has one α-helixand one β-turn (onleft upper).

Does thisrepresent an energywell?

Can you imaginea slightly higherwell?

5

3. Individual weak interactions . 1/50th to 1/10th asstrong as covalent bonds, but many may be involvedin maintaining a protein’s most stable conformation. It’s possible to view the folding in equilibrium (Keq )terms.

Is the effect of each individual interaction on Keq additive or multiplicative? To answer this remember therelationship between Keq and ΔGE :

(!ΔGE'RT)

Keq = e

6

4. Comment on ΔH vs. ΔG aspects of hydrogen bondingand maintainance of protein native conformation.

5. The “Hydrophobic Collapse” and protein folding.

6. Two guiding principles (often but not always):

a) Hydrophobic residues are usually inside the proteinb) Hydrogen bonding within the protein is maximized

7

O

CNR1

R2

H

B. Peptide bond is rigid and planar.

1. What do you predict aboutthe peptide bond from thefollowing representation?

a) orbital hybridization?b) 6 atom centers in different planes?c) H bond acceptor sites?d) Free rotation about C!N bond?

8

a) Orbital hybridization? Back to this one in a minute?b) Nope; they are all in the same plane.c) Only acceptor sites are on the carbonyl O atom.d) There is almost no rotation about the C!N bond.

What do b) to d) tell you about orbital hybridization?

N C O?

2. The 6 atoms are coplanar, what does that mean? a) Resonance, but... with separation of charge! (Organic?)b) Limits structural possibilities. Makes life easier for us re.

understanding structure. See fig. 4-2 (a)

9

3. Back to ...'N!C!C'N!C!C'... α α

a) There is relative freedom of rotation about the singlebonds on either side of the α-carbon.

b) Comment on dihedral angles

Convention:1) Look down the axis from N-term toward C-term. 2) Heading clockwise from 0 is +E , heading counter clockwise is

!E.c) φ (for the N!Cα bond) and ψ (for the Cα!C bond) are used

to describe the state of rotation (dihedral angle).

Fig. 4-2(b)10

4. Ramachandran plots are a useful way to look atallowed values of φ and ψ. Why are some anglesnot possible? Proline??? See Fig. 4-3, p. 119.

11

II. Protein Secondary (2E) Structure

In most general terms, 2E structure refers to shortrange arrangement of backbone (!N!C!C!) atoms.

Regular 2E structure exists when φ values and ψvalues are similar over a linear stretch of residues.

The most common 2E structures we encounterwill be α-helix and β-sheet. There are others, butthere are usually only found in special proteins.

12

A. α-Helix: common 2E structure in proteins.1. Regular, repeating (φ = !60E, ψ = !45 to 50E)2. Right-handed 3. 5.4 D/turn, 3.6 residues/turn. See Fig. 4-4, p. 120.4. Intrachain Hydrogen bonding contributes ΔH.

B. Amino acid sequence affects α-helix stability.1. α-Helix stability of polyglu? pH dependence?2. Stability of lysx..glux + 3ish? (-ish is 3 or 4)3. Stability of phex..trpx + 3ish?

Look at Fig. 4-4 d) if this isn’t clear.

13

4. Helix “breakers”a) pro Why b) gly: because it’s too unconstrained. (?)

5. Five general patterns:a) adjacent R group electrostatic interaction (#1) Fig. 4-5b) adjacent R group steric hinderancec) 3ish interactions (#2-3)d) pro: rare , gly: raree) R group (+/-) stabilization of helix dipole

Aside: residues at the ends of the helix do not have bothHydrogen bond sites (carbonyl O acceptor and amide Hdonor) involve in helix related Hydrogen bonding.

14

C. β-conformation involved in β-sheet formation.

0. In the β-conformation the backbone is more extendedthan in the α-helix. Does this make sense given theφ and ψ angles listed in Table 4-1?

1. When two chains in the β-conformation are alignednear each other, 2 types of β-sheets may form(polarities NÿC termini orientations):a) parallelb) antiparallel

2. Intra(er)chain Hydrogen bonding, ΔH., Fig 4-6

15

D. β-turns are a common “defined” turn mode.1. Globular proteins have to fold back on themselves. 2. Antiparallel β-sheets, 4 helix bundles, etc.3. 4 residues accomplish a 180E turn.4. 4th residue hydrogen bonds to 1st residue in turn5. Look at Fig. 4-7 more closely. It shows 2 of the

more common types of β-turns. a) Do the standard 6 atoms that should be in the same plane

always look like they are.b) Find the pro residue. Is its peptide bond trans- or cis-?

(If this is too hard to determine with Fig. 4-7, find a β-turn on the pdb.) Does the β-turn in 1ppt look like it fitsinto either Type I or type II.

16

Few (0.05%) cis-peptide bonds occur. However, ~6% of peptide bonds at Pro are cis, and

many of these are at β-turns.

Fig. 4-8

17

E. Common 2E structures have characteristicdihedral angles & some constraints on amino acidcomposition (Gly can break the rules [Why?]).

See Fig. 4-9 pyruvate kinase (below)

F. Circular Dichroism (CD) can be used to study 2Estructure. See Fig. 4-10. Δε represents difference inabsorption between left- & right-handed circularly polarizedlight.

Δε is related to the sample’s chirality. With that in mind, do these spectra make sense?

18

III. Protein Tertiary & Quaternary StructureTwo main 3E groupings: fibrous & globular

A. Fibrous proteins are adapted for structuralfunction. (Not really as awkward as it sounds.)1. α-Keratin (intermediate filament proteins), α-helical

Where? Wool, hair, claws, nails (toe?). Strength!!!2. Collagen (structure: collagen triple helix) Where?

Connective tissue: bone matrix, cartilage, tendon. Box 4-3 re. hydroxyproline, scurvy, etc. Strength!!!

3. Fibroin (insects and spiders: silk) & more strength!4. Pictures in Figs. 4-11 & 4-14,

Fibroin chains of silk are in β-sheet conformation.Scanning EM of spider spinnerets (colorized)

19

B. Structural diversity reflects functional diversityin globular proteins. Fig. 4-15: useful perspectivefor human serum albumin, Mr = 64,500.

C. Myoglobin: 1st protein w/ crystal structuresolved at relatively high resolution (1950's). 1. 153 aa residues, 1 heme, Mr = 16,7002. 8 α-helices (>70% of aa’s) wrap around the heme3. Most hydrophobic side chains are on the interior4. All but 2 polar side chains are exterior, and hydrated (His93

is one of the Fe2+ ligands) (pdbSum: 1mbo)5. . 75% of space in the protein is occupied by atoms, atomic

density comparable to that of a solid crystal (comment onstrength of interactions and being “tied down”)

20

The average globular protein is not real holey.

Sperm whale myoglobin (pdb: 1mbo).

Yellow atoms represent the side chains of hydrophobic residuesLeu, Ile, Val, and Phe. Notice that their surfaces are largely insidethe protein.

What is the red thing?

Myoglobin contains 1 heme per molecule (Fig 4-17):

21

Aside: How is 3-D protein structure determined? X-ray crystallography and 1H nuclear magneticresonance (NMR). See Box 4-5, pp. 133-136.

1. Re. crystallography:

a) Crystalsb) Timec) “One” structure

2. R. NMR:a) Solutionb) $$$ for equipment, maintenance (N2(l))c) Multiple, overlapping structures

Comment re. highly specialized type of work?

22

D. Globular proteins: large variety of 3E structures. Note: This section differs a bit from 5th ed. to 6th ed..

1. To get started look for common structural patterns. 2. Vocabulary

a) A motif (synonym: fold): a recognizable foldingpattern involving 2 or more elements of 2E structureand the connections between them.

Recognizable: visually apparent (to cognoscenti) Motifs may contain only a few components (β-α-βloop) or many (β barrels).

b) A domain: part of a polypeptide chain that isindependently stable and/or could move as an entityrelative to the rest of the chain.

23

Fig 4-18, examples of motifs

Fig. 4-19, Troponin CTroponin C (pdb: 4tnc) See next page.

Ca2+ ion shown in gray.

1) What type of structure bridges the 2 domains?

2) Can you use the information if Fig 4-4 to estimate the length ofthis structure?

24

25

E. Protein motifs are the basis for protein structureclassification. See Fig. 4-22, p. 139-140.

1. Levels of structural classification: a) class (see below) is purely structuralb) fold is purely structuralc) superfamily members: similar in major structural motif &

function, but relatively limited sequence homologyd) family members: similar 3D structure & (often) function,

& also have strong sequence homology (they’re related)e) protein f) species

e) and f) shouldn’t need much explanation.

26

2. There are 4 classesa) All α 1bcfb) All β 1pexc) α'β Means α & β regions are interspersed (in 3D?) 1pfkd) α + β Means α & β regions somewhat segregated 1ema

Closing comments: Folds usually defined by similarity to an archetype or structure

(2E element, striking geometry, etc.)Superfamily usually named by similarity to archetype, but not

always related by sequence (convergent evolution?)

27

F. Protein 4E structures: dimers to massivelycomplex multimers.

1. Terminology: protomers ÿ multimer

2. Symmetry a) rotational (within plane)b) helical (rotate and move to new plane)

3. Examples:a) Insulin: A (21 aa) & B (30 aa) chains (history?)b) Hemoglobin: α2, β2 (both with ~145 res)c) Viral capsids (eg. TMV, 2,130 identical subunits)

There are limits to protein size re. errors in translation.28

The hemoglobin (Hb) tetramer. Most students find it isway better to go to the PDB or PDBSum to view thestructure in three dimensional viewing mode. Theaccession code for this structure is 2hhb.

G. Some proteins (or sections of them) areintrinsically disordered. (Not in 5th ed.?)1. Some proteins don’t readily crystallize. Why?

These proteins don’t have just one conformation.2. Perhaps 1'3 of human proteins have unstructured

segments. (Not necessarily the whole protein.)3. These proteins tend to lack a hydrophobic core & are

rich in Lys, Arg, and Glu.

29

4. An important example is found in the N– and C-terminal regions of p53 (oncogenes?). p53 performsa number of functions associated with cell division,apoptosis, and DNA repair.a) C-terminal end of p53 must interact with numerous other

proteins to perform its varied functions. b) These other proteins have different quite different shapes

in their p53 binding regions.c) One obvious way for p53 to be able to interact with these

different shapes is to be able to change the shape of itsbinding (C-terminal) region.

See Fig. 4-24, next page. Note: PONDR: Predictor ofNatural Disorder regions, 1.0 = very likely disordered.

30

IV. Protein Denaturation and Folding

A. Loss of protein structure results in loss(change?) of protein function.

1. Proteostatsis: maintainance of active proteinsnecessary for a cell to stay alive. See Fig 4-25, nextpage.

2. Heat and/or detergent re. loss of activity.Comment on 2nd degree burns.See Fig 4-26, second page following.

31

B. AA sequence determines 3E structure.

1. Anfinsen’s studies w/ ribonuclease Fig. 4-27, p. 145.2. Comment on size and refolding

C. Polypeptides fold rapidly by stepwiseprocesses.1. Fig. 4-28, p. 145, next page.

a) Why does the bend at the bottom occur? (See res. 1-20)b) Why does the α-helix form? (Res. 28-39)

This is better animated, so see also videos:Trp-cage miniprotein folding:

http://www.youtube.com/watch?v=BBWMhtlkfeIProtein GB1 folding:

http://www.youtube.com/watch?v=meNEUTn9Atg&feature=related Why all the jiggling?

32

2. Fig. 4-29 Back to ΔGE

What gets you (the protein) get out of a valley [in (b) &(d)] over the peaks, and into the next valley?

3. Comment re. folding during synthesis as opposed tofolding after completion of synthesis.

D. Some proteins undergo assisted folding.

1. Chaperones are proteins that interact with un- orpartially folded proteins and catalyze their shift to“native” conformations. Figs. 4-30 (& 31)

33

2. Note ATP hydrolysis

3. Good thing for Anfinsen that ribonuclease didn’t achaperone?

E. Defects in protein folding provide the molecularbasis for a wide range of human genetic (andother [BSE?]?) disorders. (Change from 5th ed.)

1. Examples of health problems associated with proteinfolding: type 2 diabetes, Alzheimer disease,Huntington disease, and Parkinson’s.

2. BSE and prion misfolding, Box 4-6. 34