1. Protein Structure & Modeling Biology 224 Instructor: Tom Peavy Nov 18 & 23, 2009 <Images adapted from Bioinformatics and Functional Genomics by Jonathan Pevsner>

2. Classical structural biology Determine biochemical activity Purify protein Determine structure Understand mechanism, function

3. Structural genomics Determine genomic DNA sequence Predict protein Determine structure or analyze in silico Understand mechanism, function

4. Protein function and structure Function is often assigned based on homology. However, homology based on sequence identity may be subtle. Consider RBP and OBP: these are true homologs (they are both lipocalins, sharing the GXW motif). But they are distant relatives, and do not share significant amino acid identity in a pairwise alignment. Protein structure evolves more slowly than primary amino acid sequence. RBP and OBP share highly similar three dimensional structures.

5. Principles of protein structure Primary amino acid sequence Secondary structure:  helices,  sheets Tertiary structure: from X-ray, NMR Quaternary structure: multiple subunits

6. Protein secondary structure Protein secondary structure is determined by the amino acid side chains. Myoglobin is an example of a protein having many  -helices. These are formed by amino acid stretches 4-40 residues in length. Thioredoxin from E. coli is an example of a protein with many b sheets, formed from  strands composed of 5-10 residues. They are arranged in parallel or antiparallel orientations.

7. Myoglobin (John Kendrew, 1958)

8. Thioredoxin

9. Secondary structure prediction Chou and Fasman (1974) developed an algorithm based on the frequencies of amino acids found in  helices,  -sheets, and turns. Proline: occurs at turns, but not in  helices. GOR (Garnier, Osguthorpe, Robson): related algorithm Modern algorithms: use multiple sequence alignments and achieve higher success rate (about 70-75%)

10. Secondary structure prediction Web servers: GOR4 Jpred NNPREDICT PHD Predator PredictProtein PSIPRED SAM-T99sec

13. Tertiary protein structure: protein folding Three main approaches: [1] experimental determination (X-ray crystallography, NMR) [2] Comparative modeling (based on homology) [3] Ab initio (de novo) prediction

14. Experimental approaches to protein structure [1] X-ray crystallography -- Used to determine 80% of structures -- Requires high protein concentration -- Requires crystals -- Able to trace amino acid side chains -- Earliest structure solved was myoglobin [2] NMR -- Magnetic field applied to proteins in solution -- Largest structures: 350 amino acids (40 kD) -- Does not require crystallization

19. Access to PDB through NCBI Molecular Modeling DataBase (MMDB) Cn3D (“see in 3D” or three dimensions): structure visualization software Vector Alignment Search Tool (VAST): view multiple structures

26. Additional web-based sites to visualize structures Swiss-PDB Viewer Chime RasMol MICE VRML

27. Structural Classification of Proteins (SCOP) SCOP describes protein structures using a hierarchical classification scheme: Classes Folds Superfamilies (likely evolutionary relationship) Families Domains Individual PDB entries http://scop.mrc.lmb.cam.ac.uk/scop/

29. There are about >20,000 structures in PDB, and about 1 million protein sequences in SwissProt/ TrEMBL. For most proteins, structural models derive from computational biology approaches, rather than experimental methods. The most reliable method of modeling and evaluating new structures is by comparison to previously known structures. This is comparative modeling. An alternative is ab initio modeling. Approaches to predicting protein structures

30. obtain sequence (target) fold assignment comparative modeling ab initio modeling build, assess model Approaches to predicting protein structures

31. [1] Perform fold assignment (e.g. BLAST, CATH, SCOP); identify structurally conserved regions [2] Align the target (unknown protein) with the template. This is performed for >30% amino acid identity over a sufficient length [3] Build a model [4] Evaluate the model Comparative modeling of protein structures

32. Errors may occur for many reasons [1] Errors in side-chain packing [2] Distortions within correctly aligned regions [3] Errors in regions of target that do not match template [4] errors in sequence alignment [5] use of incorrect templates Errors in comparative modeling

33. Many web servers offer comparative modeling services. Examples are SWISS-MODEL (ExPASy) Predict Protein server (Columbia) WHAT IF (CMBI, Netherlands) Comparative modeling

34. Ab initio prediction can be performed when a protein has no detectable homologs. Protein folding is modeled based on global free-energy minimum estimates. The “Rosetta Stone” methods was applied to sequence families lacking known structures. For 80 of 131 proteins, one of the top five ranked models successfully predicted the structure within 6.0 Å RMSD (Bonneau et al., 2002). Ab initio protein structure prediction