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Workshop in Computational Structural Biology 2016 81813, 4 credit points Orly Marcu & Emma-joy Dodson Contents by Prof. Ora Schueler-Furman Introduction – When, Where, How? • When & Where: – Thursdays, Givat Ram – Lecture & Exercise: 14:0018:00, Sprinzak computer class #2 – Lectures & exercises available on moodle2 http://moodle2.cs.huji.ac.il/nu15/ course/view.php?id=81813 • How: – Make sure you have an account in CS ✓ • Exercises - Submit 8/11 exercises Due within 2 weeks Submit by email to [email protected] • Contact: Orly 87063 [email protected] Emma 87063 [email protected] Acknowledgements: Sources of figures and slides include slides from Branden & Tooze; some slides have been adapted from members of the Rosetta Community, especially from Jens Meiler Exercises in Pyrosetta have been adapted from teaching material by Jeff Gray What will we learn? • Structure prediction (mainly Rosetta; also I-TASSER, MODELLER): – from sequence alone to high resolution models (Ab-initio modeling) – From homologous structures to high resolution models MSKAVGIDLGTTYSC…… MSKAVGIDLGTTYSC…… || What will we learn? • Protein design – Engineering novel proteins not found in nature to fit a desired fold/function Gordon et. Al. JACS (2012). Computational Design of an α-Gliadin Peptidase What will we learn? • Protein-protein docking – achieve models of protein complexes given two monomers (Rosetta, PatchDock, PIPER, HADDOCK) • Interface analysis and design – identify interface “hotspots” (via computational alanine scanning); change protein specificity What will we learn? • Optimization techniques: – Energy Minimization; concepts and implementation in Rosetta – Monte-Carlo methods • Side chain modeling – Deterministic and heuristic methods for finding preferred side chain combinations given a certain backbone energy START conformations What we will not learn Existing protocols, out of this course’s scope: •Protein-ligand docking •Membrane proteins modeling and design •Peptide-protein docking •DNA & RNA modeling •Antibody modeling The code: 4 bases, 64 triplets, 20 amino acids 4 Hierarchies of protein structure • Anfinsen: sequence determines structure The building blocks: amino acids Special amino acids CO N C H H • The simplest aa • No sc • Very flexible bb H CO N C H2C CH2 CH2 H • Cyclic aa • sc Connects bb N • Very constrained bb Aliphatic amino acids • sc contains only carbon and hydrogen atoms • hydrophobic Amino acids with hydroxyl group Negatively charged amino acids Different size → different tendency for 2. structure Amide amino acids Positively charged amino acids • pKa 11.1 • pKa 12 • large sc Aromatic amino acids • sc contains aromatic ring Figure from Wikipedia Figure from Proteopedia Amino acids with sulfur Cystine Oxidation of Sulfur atoms creates covalent disulfide bond (S-S bond) between two cysteines Hydrogen bonding potential of amino acids Primary sequence: concatenated amino acids Formation of a peptide bond H +H N 3 C O C O- R cpk colors O - oxygen H - hydrogen N - nitrogen C - carbon The geometry of the peptide backbone The peptide bond is planar & polar: • Peptide bond length and angles do not change • Peptide dihedral angles define structure Dihedral angles Dihedral angles 1-4 define side chain • Dihedral angle: defines geometry of 4 atoms (given bond lengths and angles) From wikipedia The geometry of the peptide backbone The peptide bond is planar & polar: =180o (trans) or 0o (cis) • Peptide bond length and angles do not change • Peptide dihedral angles define structure The search for the native fold The Levinthal paradox: a 100 residue protein would require 1016 seconds to explore all possible conformations and choose the native one. Quick collapse to intermediate state, followed by accurate contacts formation Quick collapses followed by unfolding until near native state achieved Ramachandran plot All except Glycine Glycine: flexible backbone 33 Ramachandran plot 34 Secondary structure: local interactions Secondary structure – built from backbone hydrogen bonds helix • discovered 1951 by Pauling • 5-40 aa long • average: 10aa • right handed • Oi-NHi+4 : bb atoms satisfied • helix: i - i+5 • 310 helix: i - i+3 1.5Å/res Favored: Glu, Ala, Leu, Arg, Met, Lys Disfavored: Asn, Thr, Cys, Asp, Gly Frequent amino acids at the N-terminus of helices Ncap, N1, N2, N3 …….Ccap Pro Blocks the continuation of the helix by its side chain Asn, Ser Block the continuation of the helix by hydrogen bonding with the donor (NH) of N3 38 helix: dipole • binds negative charges at N-terminus Representation: helical wheel 1. buried 2. partially exposed: amphipathic helix 3. exposed 41 -sheet • Involves several regions in sequence • Residue side chains point up/down/up .. • Oi-NHj •Parallel and anti-parallel sheets Favored: Tyr, Thr, Ile, Phe, Trp Disfavored: Glu, Ala, Asp, Gly, Pro 42 Antiparallel -sheet • Parallel Hbonds • Pleated 43 Beta-hairpin Loops • Connect strands in antiparallel sheet G,N,D G G S,T 44 Parallel -sheet • less stable than antiparallel sheet • angled hbonds 45 Connecting elements of secondary structure define tertiary structure 46 Tertiary structure defines protein function Loops • connect helices and strands • at surface of molecule • more flexible • contain functional sites 48 Important bonds for protein folding and stability Dipole moments attract each other by van der Waals force (transient and very weak: 0.1-0.2 kcal.mol) Hydrophobic interaction – hydrophobic groups/ molecules tend to cluster together and shield themselves from the hydrophilic solvent Interplay of enthalpy and entropy in protein folding Formation of the aformentioned bonds contributes to the enthalpy of the system, decreasing protein enropy change in Gibbs free energy change in enthaply change in the entropic term The hydrophobic effect • A central effect in protein folding • Driven by entropy – gain of water molecules entropy Water molecules near hydrophobic elements have less freedom to form and break hydrogen bonds with neighboring waters More water molecules not in direct contact with hydrophobic elements Figures from post by Dr. Steve Mack on www.madsci.org The quaternary structure of a protein defines its biological functional unit 55 Quaternary structure: assembly of protein domains (from two distinct protein chains, or two domains in one protein sequence) Glyceraldehyde phosphate dehydrogenase: • domain 1 binds the substance to be metabolized, • domain 2 binds a cofactor 1. Introduction to Computational Structural Biology Experimental determination of protein structure: X-ray diffraction and NMR X-ray diffraction • Rotation of crystal enables recording different diffractions • Resolution measures diffraction angles; higher angle peaks higher resolution NMR (Nuclear Magnetic Resonance) NMR-active nuclei (w spins) 1H, 13C, 15N Application of magnetic field reorients spins – measure resonance between close nuclei Extract constraints & determine structure more constraints – better defined structure Experimental determination of structure X-ray crystallography NMR • Determines electron density – positions of atoms in structure • Highly accurate • Technically challenging • Depends on crystal (static; artifacts?) • Determines constraints between labeled spins • Allows measure of structure in solution Progress in experimental determination of structures 1950’s first protein structure solved by Kendrew & Perutz: sperm whale myoglobin Today: ~114,000 structures solved, most by x-ray crystallography