Protein Structure and Function

1
Disulfide Bonds

• Two cyteines in close proximity will form a
  covalent bond
• Disulfide bond disulfide bridge or dicysteine
  bond.
• Significantly stabilizes tertiary structure.

2
Determining Protein Structure

• There are O(100000) distinct proteins in the
  human proteome.
• 3D structures have been determined for 14000
  proteins from all organisms
• Includes duplicates with different ligands bound
  etc.
• Coordinates are determined by X-ray
  crystallography

3
X-Ray Crystallography

• The crystal is a mosaic of millions of copies of
  the protein.
• As much as 70 is solvent (water)!
• May take months (and a green thumb) to grow.

4
X-Ray diffraction

• Image is averagedover
• Space (many copies)
• Time (of the diffractionexperiment)

5
Electron Density Maps

• Resolution is dependent on the quality/regularity
  of the crystal
• R-factor is a measure of leftover electron
  density
• Solvent fitting
• Refinement

6
The Protein Data Bank

• http//www.rcsb.org/pdb/

ATOM 1 N ALA E 1 22.382 47.782
112.975 1.00 24.09 3APR 213 ATOM 2 CA
ALA E 1 22.957 47.648 111.613 1.00
22.40 3APR 214 ATOM 3 C ALA E 1
23.572 46.251 111.545 1.00 21.32 3APR
215 ATOM 4 O ALA E 1 23.948
45.688 112.603 1.00 21.54 3APR 216 ATOM
5 CB ALA E 1 23.932 48.787 111.380
1.00 22.79 3APR 217 ATOM 6 N GLY E
2 23.656 45.723 110.336 1.00 19.17
3APR 218 ATOM 7 CA GLY E 2 24.216
44.393 110.087 1.00 17.35 3APR 219 ATOM
8 C GLY E 2 25.653 44.308 110.579
1.00 16.49 3APR 220 ATOM 9 O GLY E
2 26.258 45.296 110.994 1.00 15.35
3APR 221 ATOM 10 N VAL E 3 26.213
43.110 110.521 1.00 16.21 3APR 222 ATOM
11 CA VAL E 3 27.594 42.879 110.975
1.00 16.02 3APR 223 ATOM 12 C VAL E
3 28.569 43.613 110.055 1.00 15.69
3APR 224 ATOM 13 O VAL E 3 28.429
43.444 108.822 1.00 16.43 3APR 225 ATOM
14 CB VAL E 3 27.834 41.363 110.979
1.00 16.66 3APR 226 ATOM 15 CG1 VAL E
3 29.259 41.013 111.404 1.00 17.35
3APR 227 ATOM 16 CG2 VAL E 3 26.811
40.649 111.850 1.00 17.03 3APR 228
7
Practical Assignment 1

• Get entry 2APR from the PDB. This is an Aspartic
  Protease structure.
• Download Rasmol or Raswin and load 2APR.
• Render the molecule as sticks with CPK coloring
  and print the image.
• Render the molecule as either a ribbons or
  cartoon image showing secondary structure.
• Rotate the molecule to show at least one beta
  sheet and one alpha helix. Print this image and
  turn it in as well.

8
The Protein Folding Problem

• Central question of molecular biologyGiven a
  particular sequence of amino acid residues
  (primary structure) what will the
  tertiary/quaternary structure of the resulting
  protein be
• Input AAVIKYGCALOutput 11 22
  backbone conformation(no side chains yet)

9
Protein Folding Biological perspective

• Central dogma Sequence specifies structure
• Denature to unfold a protein back to random
  coil configuration
• -mercaptoethanol breaks disulfide bonds
• Urea or guanidine hydrochloride denaturant
• Anfinsens experiments
• Denatured ribonuclease
• Spontaneously refolded into enzymatically active
  form
• Verified for numerous proteins

10
Folding intermediates

• Levinthals paradox Consider a 100 residue
  protein. If each residue can take only 3
  positions there are 3100 5 1047 possible
  conformations.
• If it takes 10-13s to convert from 1 structure to
  another exhaustive search would take 1.6 1027
  years!
• Folding must proceed by progressive stabilization
  of intermediates
• Molten globules most secondary structure
  formed but much less compact than native
  conformation.

11
Ideas on protein folding

• It is believed that hydrophobic collapse is a key
  driving force for protein folding
• Hydrophobic core!
• Proteins are in fact only marginally stable
• Native state is typically only 5 to 10 kcal/mole
  more stable than the unfolded form
• Many proteins help in folding
• Protein disulfide isomerase catalyzes shuffling
  of disulfide bonds
• Chaperones break up aggregates and (in theory)
  unfold misfolded proteins

12
The Hydrophobic Core

• Hemoglobin A is the protein in red blood cells
  (erythrocytes) responsible for binding oxygen.
• The mutation E6V in the chain places a
  hydrophobic Val on the surface of hemoglobin
• The resulting sticky patch causes hemoglobin S
  to agglutinate (stick together) and form fibers
  which deform the red blood cell and do not carry
  oxygen efficiently
• Sickle cell anemia was the first identified
  molecular disease

13
Sickle Cell Anemia
Sequestering hydrophobic residues in the protein
core protects proteins from hydrophobic
agglutination.
14
Computational Protein Folding

• Two key questions
• Evaluation how can we tell a correctly-folded
  protein from an incorrectly folded protein
• H-bonds
• Electrostatics
• Hydrophobic exposure
• Etc.
• Optimization once we get an evaluation
  function can we optimize it
• Simulated annealing
• EC
• Etc.

15
Evaluation of Protein Folds

• Empirical potential functions
• Residue-based spatial relationships among
  residues
• Stereochemistry-based molecular interactions
  (covalent electrostatic etc.) with coefficients
• Ab-initio potential functions
• Procheck etc.
• Full molecular dynamics
• Very computationally expensive

16
Threading Fold recognition

• Given
• Sequence IVACIVSTEYDVMKAAR
• A database of molecular coordinates
• Map the sequence onto each fold
• Evaluate
• Objective 1 improve scoring function
• Objective 2 folding

17
Fold Optimization

• Simple lattice models (HP-models)
• Two types of residues hydrophobic and polar
• 2-D or 3-D lattice
• The only force is hydrophobic collapse
• Score number of HH contacts

18
Learning from Lattice Models

• The hydrophobic zipper effect

Ken Dill 1997
19
Secondary Structure Prediction

• Easier than folding
• Current algorithms can prediction secondary
  structure with 70-80 accuracy
• Chou P.Y. Fasman G.D. (1974). Biochemistry
  13 211-222.
• Based on frequencies of occurrence of residues in
  helices and sheets
• PhD Neural network based
• Uses a multiple sequence alignment
• Rost Sander Proteins 1994 19 55-72

20
Secondary Structure Prediction
AGVGTVPMTAYGNDIQYYGQVT
A-VGIVPM-AYGQDIQY-GQVT
AG-GIIP--AYGNELQ--GQVT
AGVCTVPMTA---ELQYYG--T
AGVGTVPMTAYGNDIQYYGQVT
----hhhHHHHHHhhh--eeEE
21
A Peek at Protein Function

• Serine proteases cleave other proteins
• Catalytic Triad ASP HIS SER

22
Three Serine Proteases

• Chymotrypsin Cleaves the peptide bond on the
  carboxyl side of aromatic (ring) residues Trp
  Phe Tyr and large hydrophobic residues Met.
• Trypsin Cleaves after Lys (K) or Arg (R)
• Positive charge
• Elastase Cleaves after small residues Gly
  Ala Ser Cys

23
Specificity Binding Pocket
24
onward

• Apo-proteins and prosthetic groups
• Lab techniques for proteins
• Gels
• Xtal
• Digests
• Some computational areas of interest
• Folding
• Docking screening