Secondary and tertiary structure of proteins Josef Houser Autumn 2023 S1004 Methods for structural characterization of biomolecules Structure • “Relative” position of individual building blocks within the sample • Block definition reflects structural details: – Whole molecules – Domains – Secondary structures – Residues – Atoms – Orbitals – Elementary particles NH NH NH NH2 NH O NH2 OH O O NH NH O O OH SH Protein Linear polymer of amino acids linked by peptide bond N-terminus C-terminus primary (sequence) secondary tertiaryquaternary ADSQTSSNRAGEFSIPPNTDFRAIFFANAAE QQHIKLFIGDSQEPAAYHKLTTRDGPREATL NSGNGKIRFEVSVNGKPSATDARLAPINGK KSDGSPFTVNFGIVVSEDGHDSDYNDGIVV LQWPIG 4 Protein structure 2D1D Protein secondary structure Peptide bond – planar (angle ω is ± 180°) Backbone conformation defined by two torsion angles φ and ψ NH N NH NH2 N O NH2 OH O O N N O O OH SH H H H H 2D Ramachandran diagram a) Colored areas show sterically allowed combinations of the  and  angles. b) Observed values for all residue types except for glycine. Each point represents  and  values for an amino acid residue in a well-refined x-ray structure. c) Observed values for glycine 6 • Combination of φ and ψ angles for individual amino acids in protein • Populated in several areas (combination of angles) • Main areas labeled as α and β 2D Sterical hindrance 7 • Non-represented combinations suffer from sterical clashes • Much smaller problem for glycine – Cα atom missing wikipedia.org http://bioinformatics.org/molvis/phipsi/ 2D Bonds involved in structure stbilization • Hydrogen bond (H-bridge) • Charge-charge • Polar AA contacts • Non-polar / hydrophobic AAs • Stacking – aromatic AAs • Cysteine / cystine – sulfur-sulfur bond • Metal ions 8 2D α-helix 9 • Most frequent • Stabilized by intra-main chain hydrogen bonds 2D Other helix structures • 310 helix – “more tight” – ends of α helix, turns • π-helix – “more loose” – ends of helixes, very rare • left-hand helix – sequence dependent – proline/glycine rich – colagen 10 2D β-sheet (β-strand) Parallel 11 Antiparallel • Second main 2D structure type • Stabilized by intermain chain hydrogen bonds • Two types based on mutual orientation of neighboring chains • Antiparallel more stable than parallel 2D Turns • Several types • Various AAs number – 3 - 5 • Examples: β-turn, γ-turn 12www.cryst.bbk.ac.uk 2D Loops • Connecting two elements of 2D structure • Partially organized structures – between turns and random coil • Typically 5-16 AAs with dominantly polar residues 13 2D Y.Choietal(2013)Peerj. • Location of individual atoms in space – “heavy atoms” – C, N, O, S, (P) – hydrogens – bound molecules – hydration shell Protein terciary structure Absolute coordinates • Related to the defined origin of coordinate system [0, 0, 0] • Cartesian coordinates – x, y, z • Spherical coordinates – r, θ, φ or ρ, θ, φ • N atoms → 3N coordinates 15 x y z (x, y, z) Cartesian (r, θ, φ) Spherical φ θ r Relative coordinates • Related to previous defined point (atom) • Distance to previous atom • Angle between three atoms • Torsion angle between four atoms • For N atoms → 3N – 6 coordinates 16 A - - B rAB - C rBC θABC D rCD θBCD ωABCD E rDE θCDE ωBCDE ... rAB B ωABCD rBC rCD C D θBCD rDE etc. A θABC • Complicated hierarchy: Secondary – (Supersecondary) – Terciary • Topology (in structural biology) – mutual orientation of 2D structure elements • Motives – Folds – Domains From 2D to 3D structure S.Baskaran(2010) From 2D to 3D structure • Motifs – 2-3 elements of secondary structure combined • Folds – Combination of simple motifs • Domains – Consist of motifs/folds 18 Simple motifs 19 Helix-turn-helix β-hairpin β-α-β Complex α-motifs/folds 20 4-helix bundle 7-helix barrel Complex β-motifs/folds 21 Greek key β-meander β-barrel Complex α/β-motifs/folds 22 Rossman fold TIM-barrel Protein domains • Part of the structure with defined function (smallest functional unit) • Independent unit (at least partially) • Single-domain vs. multi-domain proteins 3D structure databases • wwPDB (http://www.wwpdb.org) RCSB PDB – Research Collaboratory for Structural Bioinformatics Protein Data Bank PDBe – Protein Data Bank Europe PDBj – Protein Data Bank Japan BMRB – Biological Magnetic Resonance Data Bank • SCOP (http://scop.mrc-lmb.cam.ac.uk/scop/) structural classification of proteins • CATH (http://www.cathdb.info/) classification of protein-domains • EMDataBank (http://www.emdatabank.org/) electron microscopy structures Formats for 3D structure files PDB (Protein Data Bank) • PDB File Format (http://www.wwpdb.org/documentation/file-format) • mmCIF File Format and PDB Exchange Dictionary • PDBML - XML File Format • Created 1976 • Fixed column position and width, capacity limitation • Still very frequent but outdated Amino acid type Residue number Coordinates Chain PDB format • Created 1990, preferred since 2014, since 2019 the only accepted for deposition • Non-context grammar, more flexible, possibility to add further information mmCIF format Amino acid type Residue number Coordinates Chain • Association of individual (protein) chains • Consisting of identical chains (homooligomers) or different chains (heterooligomers), including non-protein molecules, e.g. nucleic acids 28 Quaternary structure • Molecular structure is not static picture • Various degrees of dynamics – Atom thermal motion – Flexibility of side chains – Flexibility of backbone – Association/dissociation of subunits 29 Structure dynamics D Structure in time • Static – Thermal motion – low temperature – Short time scale – Averaging • Dynamic – Multiple structures comparison – Precision of structure determination – Dedicated methods • Fast change – Average signal – Continuum of states • Slow change – Sample heterogeneity – Separation of species or signals 31 Speed of change D + • 2D structure enables formation of higher structures (3D, 4D) • Experimentaly (CD, IR) determined average • Environment/interaction-induced 2D structure change 32 2D structure dynamics D • Flexible backbone – intrinsically disordered proteins (IDPs) • Flexible parts – loops, N- and C- termini – Random movement – Stabilization by ligand binding • Side chains – Preferred conformations – Stabilization by additional bonds (H, polar, hydrophobic) 33 3D structure dynamics D • Association/dissociation equilibrium • Multi-component complex – various dissociation constants 34 4D structure dynamics D concentration KD Heterogenous mixture Complex Individual subunits Visualization of 3D structures • Many SW tools: Mol*, PyMol, Jmol, LiteMol, RasMol, VMD, Chimera, Cn3D,… • Various applications (web-based, high-resolution images, platform-specific) • Several styles for different purposes Visualization of 3D structures • Spheres / Surface / Mesh • Space filling, interaction surfaces, overall shape Visualization of 3D structures • Cartoon / Ribbon • Secondary structures, domain assignment, connectivity, main chain orientation Visualization of 3D structures • Sticks / Balls & sticks / Lines • Detailed view, side chain orientation, mutations Visualization of 3D structures • Combination of representations – for publication purposes • Additional graphics – hydrogen bonds, distances, clashes, labeling, electron density, ... Good figure should be both nice and clear (!) Questions? 40 ? CF Head Josef Houser • +420 549 492 527 • josef.houser@ceitec.cz bic@ceitec.cz bic.ceitec.cz Biomolecular I nteractions and Crystallography Core Facility