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
6 The 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 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
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
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
Optimization once we get an evaluation function can we optimize it
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
Full molecular dynamics
Very computationally expensive
16 Threading Fold recognition
A database of molecular coordinates
Map the sequence onto each fold
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
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)
Elastase Cleaves after small residues Gly Ala Ser Cys
23 Specificity Binding Pocket 24 onward
Apo-proteins and prosthetic groups
Lab techniques for proteins
Some computational areas of interest
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