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

Cys Cis Pro 164 184

Glu 163

4.1 Å 3.0 Å 3.2 Å 3.0 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 (main- chain and side-chain); non-covalent interactions"

• Quaternary, 4o! – 3-D arrangement of subunit chains, non-covalent interactions Four Levels of Protein Four LevelsStructure of Protein Structure

• Primary, 1o " TPEEKSAVTALWGKV" • Secondary, 2o!

• Tertiary, 3o! α2 α1

β2 • Quaternary, 4o!

β2 β1 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 H- bonds; 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 1HSA – 266L Peptide bound to • Disulfide bond Class I MHC – 203C:259C Quaternary Structure

α2 α1

β2 β1 PDB File Internal coordinates: bond lengths Inter-atomic distances:(distance between bonded atoms) Interatomic Distances

Atomic coordinates

a1 = (a1x, a1y, a1z) a2 = (a2x, a2y, a2z)

Bond length

2 2 2 1/2 b12 = ((a2x–a1x ) +(a2y–a1y ) +(a2z–a1z ) )

a2

b12

a1 PDB file 3e7l Bonded distances don’t change much: Bond lengths:Bond Chain A backbone Lengths Remain Constant

1.459±0.003 Å 1.527±0.003 Å 1.329±0.001 Å Bond or “valence”Internal angles: coordinates: valence angles (complement of Bondangle formed byAngles successive chemical bonds) 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 b23 b12"b23 = b12b23 cos("–!123)

!123 b12 a3 cos!123 = –b12"b23/(b12b23)

a1 cos("–!123) = cos" cos!123 + sin" sin!123 = – cos!123 DistrDistributionibution of backbone of angles:PDBBackbone file 3e7l 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 Glu Cys Cys His His

35Å 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?