Peptides & Primary Structure: Key Concepts & Objectives for BIOC 460, Spring 2008, Study notes of Biology

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BIOC 460, Spring 2008 LEC 4, Peptides/Primary Structure with Key Concepts and Learning Objectives 1 LEC 4, Peptides and Primary Structure: Key Concepts • Proteins: primary structure – Peptide bond • amide linkage holding amino acid residues in peptide and protein polymers (primary structure of proteins). • Product of condensation of 2 amino acids – Posttranslational modifications of amino acids/proteins Examples: • hydroxylation of some Pro and Lys residues in collagen (vital for collagen structure) • carboxylation of some Glu residues (vital for blood clotting) • reversible phosphorylation of some Ser, Thr, and Tyr residues (vital for many regulatory processes) • proteolytic cleavage (vital for some regulatory processes and in digestion of protein nutrients) • disulfide bond formation (vital for structures of some proteins, especially extracellular proteins, and in some coenzyme and enzyme activities) – Sequence of amino acids in protein (primary structure) determines 3-dimensional folding pattern of protein (higher levels of structure). Key Concepts, continued • Properties of the peptide bond – Partial double bond character of peptide bond -- important consequences for 3-dimensional structures of proteins: • planarity of 6-atom peptide unit (peptide bond C=O and N-H in center, plus αCs on both sides of peptide bond) • no free rotation (cis-trans isomerism) • steric constraints on dihedral angles around backbone bonds for each amino acid residue – N-Cα angle: Φ – Cα-C=O angle: Ψ • Ramachandran diagram: plot of Ψ vs. Φ (angular coordinates) of amino acid residues in protein(s) BIOC 460, Spring 2008 LEC 4, Peptides/Primary Structure with Key Concepts and Learning Objectives 2 Learning Objectives (See also posted Peptide/pH/Ionization practice problems.) • Terminology related to polypeptides: amino acid residue, backbone, side chains, disulfide bonds, conformation, configuration • Write the chemical equation for formation of a peptide bond. • Draw a peptide bond and describe its conformation (3-dimensional arrangement of atoms). • Explain the relation between the N- and C-terminal residues of a peptide or protein and the numbering of the amino acid residues in the chain, and be able to draw a linear projection structure (like text Fig. 2.19) of a short peptide of any given sequence, using the convention for writing sequences left to right from amino to carboxy terminus. • Be able to estimate the approximate net charge on a short peptide at any given pH. This requires being given or knowing the approximate pKa values of the ionizable groups in peptides and proteins (the single α-amino group and single α-carboxyl group on the peptide, and any ionizable R groups) as well as the chemistry/charge properties of those groups in their conjugate acid and conjugate base forms. Learning Objectives, continued • Explain how the partial double bond character of the peptide bond and steric effects relate to the conformation of a polypeptide chain, including whether peptide bonds in proteins are predominantly cis or trans. • Explain the concept of a 6-atom planar peptide group (from one α-C to the next α-C), and how one plane can rotate relative to the next plane in a polypeptide backbone, around the Φ and/or Ψ angles. • Explain which bond rotation angle is defined/described as Φ and which bond rotation angle is described as Ψ. Explain what a Ramachandran plot is, and how it relates to "allowed" combinations of (Φ,Ψ) coordinates for proteins. BIOC 460, Spring 2008 LEC 4, Peptides/Primary Structure with Key Concepts and Learning Objectives 5 Backbone of peptide • Hydrogen bonding potential • Nomenclature: L-aspartyl-L-phenylalanine methyl ester (Asp-Phe-O- CH3) -- common names? • Oligo- vs. Polypeptide • Mass in daltons (amu) or kilodaltons (kD) • Mean residue weight ~110 daltons Posttranslational modification of proteins • Chemical modification after protein synthesis • Modification carried out by specific enzymes • Examples: – Hydroxylation of Pro or Lys • 4-hydroxyproline (Hyp) • Enzyme: prolyl hydroxylase • Collagen/connective tissue • Vitamin C required for the hydroxylase • What disease results from vitamin C deficiency? BIOC 460, Spring 2008 LEC 4, Peptides/Primary Structure with Key Concepts and Learning Objectives 6 Posttranslational modification of proteins • Carboxylation of specific Glu residues on gamma (γ) carbon • Enzyme: glutamate carboxylase • γ-carboxyGlu (gla) • Required for function of several blood clotting enzymes • Involved in Ca2+ binding/ co-localization with platelets at wound sites • Vitamin K required for recycling active form of carboxylase) What would be the effect of a deficiency in Vitamin K? Posttranslational modification of proteins • Phosphorylation of specific Ser (S), Thr (T), or Tyr (Y) residues by (enzymes): protein kinases (add PO32–) Phospho-Ser Phospho-Tyr • Modification removed by (different enzymes): protein phosphatases (remove PO32–) What type of bond links the phosphate to the Ser or Thr or Tyr side chain? BIOC 460, Spring 2008 LEC 4, Peptides/Primary Structure with Key Concepts and Learning Objectives 7 Disulfide Bond Formation (oxidation of cysteinyl residues) • --> covalent crosslinks between Cys residues • Oxidation (loss of 2 e–) makes diS bond. • Reduction (gain of 2 e–) breaks diS bond. Berg et al., Fig. 2.21 Amino acid sequence (primary structure) • Sequence (order) of amino acids in chain • Product of translation on a ribosome (and subsequent postranslational modifications) •Information flow: DNA --> RNA --> AA sequence --> 3-D folded protein structure BIOC 460, Spring 2008 LEC 4, Peptides/Primary Structure with Key Concepts and Learning Objectives 10 Steric constraints favor trans config. • Dihedral angles, on either side of each α C • Planar units (from α C of one residue to α C of next residue) rotate around 2 bonds: Berg et al., Fig. 2-27 Phi (Φ) (N–Cα) Psi (Ψ) (Cα–CO) phi-psi animation • Backbone of chain folds in 3 dimensions. • Each A.A. residue in 3-D structure of protein has its own (Ψ,Φ) coordinates around its αC, which determine orientation of that α C relative to preceding and following αC's. • About 3/4 of (Ψ,Φ) coordinates/combinations not allowed (forbidden by steric constraints) 4 successive planar peptide groups bounded by the α carbons of 5 successive amino acid residues BIOC 460, Spring 2008 LEC 4, Peptides/Primary Structure with Key Concepts and Learning Objectives 11 Ramachandran diagram (Ψ vs. Φ plot) • Allowed (Φ,Ψ) combinations depend on local sequence (R groups) • Shaded to show allowed conformations for non-glycine residues Berg et al., Fig. 2-28 Terminology • Conformation: spatial arrangement of atoms/groups that can change by bond rotation with no covalent bond breaking • Configuration: spatial arrangement of atoms/groups that cannot change without breaking covalent bonds • Protein Secondary Structures (next lecture) • Properties of peptide bond and hydrogen bonding favor specific kinds of repetitive local structures in proteins like – α-helix – β-conformation