BIOC 460 Summer 2010
Peptides and Protein Primary Structure Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 2, pp. 34-37 Work on Peptide Ionization Practice Problems on Lecture Notes page. Problems in textbook: chapter 2, pp. 63-64, #6,7,8,13,14 Molecular Graphics Routines: Jmol structure showing planarity of peptide bond: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/peptide/peptide.html
Animation of phi (Φ) and psi (Ψ) angles (dihedral angles for an amino acid residue): http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Structure/Rama_animationhtm.htm Two cool protein synthesis animations: View this one first: http://www.youtube.com/watch?v=PEDQoQuIhkg&feature=related A more “real” view: http://www.youtube.com/watch?v=X9vIOYlZXjE 1
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 3dimensional folding pattern of protein (higher levels of structure). 2
Peptides/Primary Structure
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BIOC 460 Summer 2010
Key Concepts, cont’d • 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 α angle: φ – Cα α-C=O angle: ψ • Ramachandran diagram: plot of φ vs. ψ(angular coordinates) of amino acid residues in protein(s) 3
PROTEINS: PRIMARY STRUCTURE (AMINO ACID SEQUENCE) •
Sequence (order) of amino acids in chain
•
Product of transcription from genomic DNA to mRNA then translation on a ribosome (and subsequent postranslational modifications) to make protein
•Information flow: DNA --> RNA --> AA sequence --> 3-D folded protein structure
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Peptides/Primary Structure
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BIOC 460 Summer 2010
Amino acid sequence (primary structure) • Fig. 2.18: Peptide bond formation. condensation of carboxylic acid and amino group
What kind of bond/linkage is a peptide bond? (Ester, anhydride, ether, amide, …?) 5
• In 55.5 M H2O, will equilibrium lie in the direction of peptide bond formation or hydrolysis? • Why aren’t we all just puddles of amino acids?
• Thermodynamics (direction) vs. Kinetics (rate)
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Peptides/Primary Structure
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BIOC 460 Summer 2010
Terminology: N-terminus, C-terminus, A.A. residue Numbering Backbone Polarity Sequence Composition
N
C
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Backbone of peptide α
α
α
α
α
• Which atoms in the peptide backbone are tetrahedral? • Hydrogen bonding potential • Oligo- vs. Polypeptide • Mass in daltons (amu) or kilodaltons (kD) • Mean residue weight ~110 daltons 8
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BIOC 460 Summer 2010
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? 9
Posttranslational modification of proteins • Carboxylation of specific Glu residues on gamma (γγ) 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?
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BIOC 460 Summer 2010
Posttranslational modification of proteins • Phosphorylation of specific Ser (S), Thr (T), or Tyr (Y) residues by (enzymes): protein kinases (add PO32–)
What type of bond links the phosphate to the Ser or Thr or Tyr side chain? Phospho-Ser Phospho-Tyr • Modification removed by (different enzymes): protein phosphatases (remove PO32–)
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• 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
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BIOC 460 Summer 2010
Properties of the peptide bond • Partial double bond character --> resonance structures – Consequences: •Planarity (6 coplanar atoms) •Rigidity (no free rotation) •Cis-trans isomers of peptide bond possible •Dihedral angles, Φ and Ψ
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Planarity of Peptide bond • 6 coplanar atom animation: http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/peptide/peptide.html
Cα(n)–CO–NH–Cα(n+1)
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Berg et al. Fig. 2-23
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BIOC 460 Summer 2010
Peptide Bond: Cis-Trans Isomerization • Rigidity - no free rotation (partial d.b. character) α carbons on same (cis) or opposite (trans) sides of “double” bond (peptide bond) • Trans configuration highly favored (steric)
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Berg et al., Fig. 2-24
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:
Phi (Φ Φ ) (N–Cα)
Psi (Ψ Ψ) (Cα–CO)
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Structure/Rama_animationhtm.htm
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BIOC 460 Summer 2010
4 successive planar peptide groups bounded by the α carbons of 5 successive amino acid residues
• Backbone of chain folds in 3 dimensions. • Each A.A. residue in 3-D structure of protein has its own (Φ,Ψ) coordinates around its αC, which determine orientation of that αC relative to preceding and following αC's. • About 3/4 of (Φ, Ψ) coordinates/combinations not allowed 17 (forbidden by steric constraints)
Ramachandran diagram (φ φ vs ψ plot)
Berg et al., Fig. 2-28
• Shaded to show allowed conformations for non-glycine residues • Allowed (Φ,Ψ Φ,Ψ) Φ,Ψ combinations depend on local sequence 18 (R groups)
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BIOC 460 Summer 2010
φ,ψ: φ,ψ What’s it all about? • Polypeptides are made from amino acids. • Each amino acid has a R-group. • Each R-group has its own chemical character (steric volume and shape, hydrophobicity, hydrophilicity, charge, etc.) • Adjacent R-groups interact with each other. • This interaction leads to the discrete φ,ψ ψ angles about each alpha carbon. • The collective effect of these φ,ψ angles gives rise to secondary structure:α-helix and β-conformation
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Learning Objectives 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 (3dimensional 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. (Concepts discussed in Lec. 3). • 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.
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Peptides/Primary Structure
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BIOC 460 Summer 2010
Learning Objectives, continued • 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 how rotation around φ and ψ is constrained and how that is related to a Ramachandran plot for the coordinates for proteins.
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