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LEC4_Peptides
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Lecture 4: Peptides and Protein Primary Structure [PDF]
Reading: Berg, Tymoczko & Stryer, Chapter 2, pp. 34-37
Practice problems (peptide ionization) [PDF]; problems in textbook: chapter 2, pp. 63-64, #6,7,8,13,14
Updated on: 1/18/07 at 3:30 pm
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, including:
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)
Properties of the peptide bond
Partial double bond character of peptide bond -- important consequences for 3-D 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)
Objectives
See also posted Peptide/pH/Ionization practice problems.
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 pK a 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.
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.
Define the following terms as they relate to polypeptides: amino acid residue, main chain, side chains, disulfide bonds, conformation, configuration.
PROTEINS: PRIMARY STRUCTURE (AMINO ACID SEQUENCE)
biosynthesized on ribosomes
complex molecular "machines" that link amino acids
Condensation of α-carboxyl group of 1 amino acid with the α-amino group of another
forms amide linkage = peptide bond
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Berg, Tymoczko & Stryer Fig. 2.18: Peptide bond formation.
Condensation direction (left to right) is accompanied by loss of H2O; reverse direction is peptide bond hydrolysis.
Equilibrium in 55.5 M H2O lies far to the left (hydrolysis direction)
Protein synthesis (peptide bond formation) requires input of free energy to drive reaction in unfavorable direction (condensation).
Peptide bond kinetically stable: in absence of a catalyst for hydrolysis reaction, even in 55.5 M H2O, lifetime of peptide bond much longer than biological
lifetimes
"polarity" of chain (= "directionality", totally different use of the word from chemical terminology):
free α-amino group on the first amino acid residue in the chain and a free α-carboxyl group on the last residue in the chain
amino acid "residues" in peptide chains (each residue in chain smaller than free amino acid by mass of "lost" elements of H2O)
amino acid residues numbered from amino end of chain toward carboxyl terminus
starting at the amino end of the chain, ending at the carboxyl terminus
convention: sequence written left to right from "N-terminus" to "C-terminus"
Berg, Tymoczko & Stryer Fig. 2.19: Sample sequence of amino acids (Note POLARITY of backbone.)
BACKBONE of chain ("main chain") repeats down the chain:
...-N-Cα1 -C(O)-N-Cα2 -C(O)-N-Cα3 -C(O)-... etc.
sequence differences due to which R group is attached to each C α (20 different amino acids to choose from).
different proteins (different sequences) --> different amino acid compositions
Berg, Tymoczko & Stryer Fig. 2.20: Components of a polypeptide chain (backbone in black, side chains/R groups in green)
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Note hydrogen-bonding potential of amide N–H and C=O backbone groups
Backbone groups' hydrogen bonds very important in stabilizing secondary structures
Terminology:
"peptides" (or "oligopeptides", where "oligo" means "a few"):
short polymers (< ~50 AA residues)
some biologically important peptides:
peptide hormones (e.g., insulin, glucagon, oxytocin, enkephalins…)some antibiotics (e.g., gramicidin S -- has cyclic
structure so no terminal amino & carboxyl groups), includes 2 residues of D-Phe.
Commercially important peptide: L-aspartyl-L-phenylalanine methyl ester -- common names?
"Polypeptide" chains (single chains of proteins) generally 50-1000 or 2000 residues long
average MW among the 20 amino acids is about 128, minus 18 (MW of H2O) --> mean MW of each a.a. residue ~110
polypeptide chain MWs generally ~5500-220,000
MW has no units
proteins often described by mass
protein mass units = daltons, where 1 dalton = 1 atomic mass unit (~the mass of an H atom) or 1 kilodalton
(kd) = 1000 atomic mass unit
Posttranslational modification of proteins (See also Berg, Tymoczko & Stryer pp. 69-70, with Fig. 3.59)
Some proteins have some amino acid residues chemically modified after synthesis.
Modification reactions are catalyzed by specific enzymes. (Modifications sometimes are removed by OTHER specific enzymes.)
Examples:
Hydroxylation of proline or lysine residues (e.g., in collagen)
hydroxyproline (Hyp)
hydroxylating enzyme requires ascorbic acid for activity, so vitamin C deficiency causes structural abnormalities in
collagen (scurvy).
Carboxylation of specific glutamate residues (in blood clotting enzymes)
γ-carboxyglutamate
γ-carboxyGlu residues involved in Ca2+ binding
Vitamin K deficiency --> insufficient γ-carboxyglutamate in clotting enzymes, thus to defective blood clotting
(hemorrhage).
Acetylation of N-terminal α-amino group --> α-N-acetyl protein (amide linkage)
protection against degradation of protein
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Phosphorylation of specific serine, threonine, or tyrosine residues (reversible)
Protein KINASES catalyze phosphorylation, protein PHOSPHATASES catalyze DEphosphorylation.
VERY important in regulation of enzyme activities, and in signal transduction
O-phosphoserine
O-phosphotyrosine
Glycosylation (addition of carbohydrate units) on specific serine, threonine, or asparagine residues
esp. on secreted proteins and parts of proteins exposed on outer surfaces of cell membranesmakes protein more
hydrophilic
provides recognition (binding) sites for interaction with other proteins
Fatty acylation of α-amino group (amide linkage) or of specific cysteine residues (thioester linkage)
makes protein more hydrophobic
sometimes helps tether protein to membrane
ADP-ribosylation (addition of an ADP-ribosyl group)
diphtheria toxin catalyzes ADP-ribosylation of elongation factor 2 (involved in eukaryotic protein synthesis)
inactivates the elongation factor
cholera toxin catalyzes ADP-ribosylation of a specific G-protein involved in a signaling pathway
inactivates GTPase enzyme activity of G-protein --> G-protein action cannot be "turned off"
Effects of irreversibly "turned on" G-protein can lead to massive water loss and death from dehydration.
Proteolytic cleavage (hydrolysis) of polypeptide chains at specific places (peptide bonds) in the amino acid sequence
converts inactive forms to biologically active proteins
just a few examples:
digestive enzymesblood clotting enzymesHIV proteasehormones like insulin and growth hormone
caspases (important in programmed cell death, apoptosis)
disulfide bond formation (oxidation of cysteinyl residues) --> covalent crosslinks between Cys residues in different parts of
same chain or different polypeptide chains in same protein.
Berg, Tymoczko & Stryer Fig. 3.21: Disulfide bond formation.
Formation of -S-S- bond requires oxidation of 2 -SH groups (thiols, sulfydryl groups)
oxidation = loss of 2 electrons, which are accepted by another compound, unspecified in this figure
reduction (gaining of 2 electrons donated by another compound) breaks the disulfide bond
Berg, Tymoczko & Stryer Fig. 2.21: Disulfide bonds form cross-links between Cys residues on either the same
polypeptide chain OR on different chains.
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Amino acid sequence (primary structure):
sequence of amino acids in a protein synthesized on ribosomes is genetically determined.
process = "translation"
Fred Sanger: Nobel Prize for first demonstration that each polypeptide chain has a unique, precisely defined AA sequence (dictated by
the DNA sequence of the gene encoding that polypeptide)
determined AA sequence of bovine insulin, a protein hormone
Berg, Tymoczko & Stryer Fig. 2.22: Amino acid sequence of bovine insulin
Insulin posttranslationally modified, cleaving it --> 2 chains connected by disulfide bonds
mutation in gene can change amino acid residue in protein
sometimes such changes in primary structure (sequence) --> extremely deleterious effects on function of protein
severe or even fatal genetic diseases (e.g. cystic fibrosis; phenylketonuria; hemophilia; sickle cell anemia….)
AA sequence (primary structure of protein) determines 3-dimensional structure of polypeptide chain
chain folds during/after biosynthesis --> 2° and 3° structure dictated by 1° structure
subunits (individual chains) in multisubunit proteins assemble based on their 3° structures
Properties of the peptide bond
PARTIAL DOUBLE BOND CHARACTER (resonance structures)
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Consequences of partial double bond character of peptide bond:
planarity: 6 atoms in planar unit
Cα(n) –CO–NH–Cα(n+1)
Jmol structure of the 6 coplanar atoms in peptide bond
Berg, Tymoczko & Stryer Fig. 2.23: Planarity of the peptide bond -- 6 coplanar atoms:
Cα(n) –CO–NH–C α(n+1)
Side chains (R groups) shown as green balls.
rigidity (no free rotation around the peptide bond.)
cis-trans isomerism
2 possible configurations: α-carbons being on same or opposite sides of double bond
Berg, Tymoczko & Stryer Fig. 2.25: Trans and cis peptide bonds
STERIC CONSTRAINTS
favor TRANS configuration
α-carbons with attached R groups on diagonally opposite "corners" of 6-atom plane with peptide bond in the center
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> 99% of peptide bonds in proteins are trans
Exception: X–Pro peptide bonds, ~10% of which are cis
due to the constraints in ring structure of Pro’s R-group attached to amide N
angles of rotation around N–Cα bond and Cα–CO bond limited in range to avoid steric hindrance (2 atoms trying to occupy
same space)
Dihedral angles ("torsion angles") (Φ and Ψ):
α-carbons are tetrahedral, so rotation possible around two bonds in the peptide backbone for each residue:
the N–C α bond and the Cα –CO bond
rotation angles (dihedral angles, torsion angles):
Φ for the N–Cα bond
Ψ for the Cα–CO bond
3-D structure of protein: each residue has a defined set of (Ψ,Φ) angles/coordinates
determine "direction" of backbone in 3 dimensions for that residue
(Ψ,Φ) coordinates for all the residues of the protein --> entire backbone structure in 3 dimensions
Berg, Tymoczko & Stryer Fig. 2.27: Rotation about the backbone N–Cα bond and Cα–CO bond in a polypeptide (the
dihedral angles, Φ and Ψ)
steric clashes between backbone atoms and side chain atoms with certain combinations of Φ and Ψ angles, so some
combinations not "allowed"
animation: phi/psi rotation
Each amino acid residue in protein has (Ψ,Φ) coordinates describing 3-dimensional orientation of backbone of that residue.
Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed. (2004), Fig. 4-2b
Berg, Tymoczko & Stryer: Fig. 2.28: Ramachandran diagram (Ψ vs. Φ plot):
What's allowed and what's not sterically possible obviously depends partially on the nature of R groups on adjacent
residues, so depends on local sequence.
"Allowed" (Ψ,Φ) coordinates for most local sequences shown in dark green; "borderline" combinations shown in light green;
combinations generally not allowed in colorless regions of (Ψ,Φ) space.
Steric exclusion means that about 3/4 of all (Ψ,Φ) combinations (spatial arrangements) for individual residues are
"forbidden".
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Terminology:
Conformation: spatial arrangement of atoms/groups that changes by bond rotation without breaking covalent bonds.
Configuration: spatial arrangement of atoms/groups that cannot be changed without breaking covalent bonds.
Review before discussion of secondary structure of proteins: Properties of hydrogen bonds
Strongest are LINEAR.
Groups capable of forming hydrogen bonds tend to do so to the maximum extent; this includes the peptide bond’s N–H (hydrogen
bond donor) and C=O (hydrogen bond acceptor).
PROPERTIES OF PEPTIDE BOND AND HYDROGEN BOND FAVOR CERTAIN KINDS OF REPETITIVE LOCAL STRUCTURES IN
PROTEINS, SECONDARY STRUCTURES SUCH AS THE α-HELIX AND β-CONFORMATION.
[email protected] .edu
Department of Biochemistry & Molecular Biophysics
The University of Arizona
Copyright (©) 2007
All rights reserved.
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