Title: Protein Structure and Function
1Disulfide Bonds
- Two cyteines in close proximity will form a
covalent bond - Disulfide bond disulfide bridge or dicysteine
bond. - Significantly stabilizes tertiary structure.
2Determining 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
3X-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.
4X-Ray diffraction
- Image is averagedover
- Space (many copies)
- Time (of the diffractionexperiment)
5Electron Density Maps
- Resolution is dependent on the quality/regularity
of the crystal - R-factor is a measure of leftover electron
density - Solvent fitting
- Refinement
6The Protein Data Bank
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
7Practical 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.
8The 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)
9Protein 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
10Folding 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.
11Ideas 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
12The 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
13Sickle Cell Anemia
Sequestering hydrophobic residues in the protein
core protects proteins from hydrophobic
agglutination.
14Computational 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.
15Evaluation 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
16Threading 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
17Fold 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
18Learning from Lattice Models
- The hydrophobic zipper effect
Ken Dill 1997
19Secondary 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
20Secondary Structure Prediction
AGVGTVPMTAYGNDIQYYGQVT
A-VGIVPM-AYGQDIQY-GQVT
AG-GIIP--AYGNELQ--GQVT
AGVCTVPMTA---ELQYYG--T
AGVGTVPMTAYGNDIQYYGQVT
----hhhHHHHHHhhh--eeEE
21A Peek at Protein Function
- Serine proteases cleave other proteins
- Catalytic Triad ASP HIS SER
22Three 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
23Specificity Binding Pocket
24onward
- Apo-proteins and prosthetic groups
- Lab techniques for proteins
- Gels
- Xtal
- Digests
- Some computational areas of interest
- Folding
- Docking screening