Download Peptides and Protein Primary Structure

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
1
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
4
Peptides/Primary Structure
2
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)
6
Peptides/Primary Structure
3
BIOC 460 Summer 2010
Terminology: N-terminus, C-terminus, A.A. residue
Numbering
Backbone
Polarity
Sequence
Composition
N
C
7
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
Peptides/Primary Structure
4
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?
10
Peptides/Primary Structure
5
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–)
11
• 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
Peptides/Primary Structure
12
6
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
13
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)
14
Berg et al. Fig. 2-23
Peptides/Primary Structure
7
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)
15
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
Peptides/Primary Structure
16
8
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)
Peptides/Primary Structure
9
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
19
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.
20
Peptides/Primary Structure
10
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.
21
Peptides/Primary Structure
11