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Principles of Protein Structure Amino Acids - Basic Building Blocks of Proteins Amino Acids Have Molecular Chirality Molecular Chirality Proteins are Polymers Cis Proline in the Active Site of HCV NS2 Cis Pro 164 Cys 184 Glu 163 3.0 Å 4.1 Å 3.0 Å 3.2 Å 3.1 Å 3.1 Å His 143 Leu 217 (C-term) Nature 2006 vol. 442 (7104) pp. 831-5 Cis trans Proline Isomerase • Cyclophilins are a family of proteins that catalyze the isomerization of peptide backbones at a proline • Critically important for protein folding • Cyclospronine is a potent immunosuppressant and an inhibitor of cyclophilins • Commonly used after organ transplant Four Levels of Protein Structure Four Levels of Protein Structure • Primary, 1o! – Amino acid sequence; Covalent bonds" • Secondary, 2o! – Local conformation of main-chain atoms (Φ and Ψ angles); non-covalent interactions (h-bonds)" • Tertiary, 3o! – 3-D arrangement of all the atoms in space (mainchain and side-chain); non-covalent interactions" • Quaternary, 4o! – 3-D arrangement of subunit chains, non-covalent interactions Four Levels of Protein Four Levels of Protein Structure Structure • Primary, 1o " TPEEKSAVTALWGKV" • Secondary, 2o! • Tertiary, 3o! • Quaternary, 4o! α2 α1 β2 β1 β2 Side Chain Conformation Protein Backbone Conformation α Helices α Helix • If N-terminus is at bottom, then all peptide N-H bonds point “down” and all peptide C=O bonds point “up”. • N-H of residue n is H-bonded to C=O of residue n+4. • a-Helix has: – 3.6 residues per turn – Rise/residue = 1.5 Å – Rise/turn = 5.4Å Secondary Structure: Alpha Helices Right handed Left handed 310 and π Helices 310 helix H-bonds n and n+3 π helices H-bonds n and n+5 α Helix • R-groups in α-helices: – extend radially from the core, – shown in helical wheel diagram. – Can have varied distributions Polar Hydrophobic Amphipathic Secondary Structure: Beta Sheet Antiparallel beta sheet Parallel beta sheet β Sheet • Stabilized by H-bonds between N-H & C=O from adjacent stretches of strands • Peptide chains are fully extended pleated shape because adjacent peptides groups can’t be coplanar. β Sheet - 2 Orientations Parallel Not optimum Hbonds; less stable Anti-parallel Optimum H-bonds; more stable Beta Turn – 2 Conformations Only Difference Tertiary Structure • Charge based interactions – 62R:163E – 55E:170R • Hydrophobic interactions – 189V – 201L – 213I – 215L – 266L • Disulfide bond – 203C:259C 1HSA Peptide bound to Class I MHC Quaternary Structure α2 α1 β2 β1 PDB File Inter-atomic Internal coordinates: bond lengths distances: (distance between bonded atoms) Interatomic Distances Atomic coordinates a1 = (a1x, a1y, a1z) a2 = (a2x, a2y, a2z) Bond length b12 = ((a2x–a1x )2+(a2y–a1y )2 +(a2z–a1z )2)1/2 a2 b12 a1 PDB file 3e7l Bonded distances don’t change much: Bond Lengths Remain Constant lengths: Chain A backbone 1.459±0.003 Å 1.527±0.003 Å 1.329±0.001 Å Bond or “valence” angles: Internal coordinates: valence angles (complement of angle formed by successive chemical bonds) Bond Angles Atomic coordinates a1 = (a1x, a1y, a1z) a2 = (a2x, a2y, a2z) a3 = (a3x, a3y, a3z) Bond vectors b12 = (a2x–a1x, a2y–a1y, a2z–a1z) b23 = (a3x–a2x, a3y–a2y, a3z–a2z) Scalar product b12"b23 = (a2x–a1x)(a3x–a2x)+(a2y–a1y)(a3y–a2y)+(a2z–a1z)(a3z–a2z) a2 b12 a1 !123 b23 b12"b23 = b12b23 cos("–!123) a3 cos!123 = –b12"b23/(b12b23) cos("–!123) = cos" cos!123 + sin" sin!123 = – cos!123 Distribution of Backbone Angles PDB file 3e7l Distribution of backbone angles: Valence angles: Chain A backbone 112.0±1.5° 116.4±0.5° 121.7±0.7° Distribution of B-factors Motifs, Topologies and Folds: β Sheet The arrangement of secondary structure elements that give rise to a folded entity Motifs, Topologies and Folds: β Sheet Motifs, Topologies and Folds: βαβ Motifs, Topologies and Folds: α-helical Motifs, Topologies and Folds: α-helical C N Middle Domain of eIF4G - scaffold protein for translation initiation factors. Protein Domains • A folded unit of protein • normally formed around a hydrophobic core • Tend to be globular • 150-250 amino acids Protein Domains Many Eukaryotic proteins consist of multiple domains • Limited proteolysis can be used to experimentally determine domain limits. Limited Trypsin Digestion in the Absence and Presence of dsRNA Domain Swapping Location of His 143, Glu 163, Cys 184 Glu His Cys Cys 35Å Glu His HIV Protease Retroviral aspartic protease - dimerization forms a single active site Structural Comparison • Structural comparison requires a 3-Dimension search • Minimize the search by using just secondary structural elements - Potential problem? • DALI server http://ekhidna.biocenter.helsinki.fi/dali_server/ • PDBe/fold http://www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ ssmserver • VAST http://www.ncbi.nlm.nih.gov/Structure/VAST/ SCOP:Structural Classification Of Proteins SCOP database classifies domains not entire proteins CATH:Class, Architecture, Topology, Homologous Superfamily How to classify proteins with same general arrangement of secondary structure but are connected differently?