Download Proteins are polymers consisting of amino acids linked by peptide

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Proteins are polymers consisting of amino acids
linked by peptide bonds
Proteins contain a wide range of functional
groups
Proteins
P
i can interact
i
with
i h one another
h and
d with
ih
other biological molecule
The function of a protein depend on its 3dimensional shape
The amino acid sequence of a protein
determines the 3D shape of a protein
Each amino acid consist of
• a central carbon atom
• an amino group, NH2
• a carboxyl group, COOH
• a side chain
Amino acid side chains vary
in shape, size, polarity and charge
http://www.ecosci.jp/amino/amino2j_e.html
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Aliphatic
Aromatic
Nonpolar
Polar
Charged
Ala
Tyr
Gly
Asn
Asp
Val
Trp
Cys
Gln
Glu
Leu
Phe
Pro
Ser
Lys
Ile
His
Met
Thr
Arg
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Resonance structure:
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The properties of the peptide bond have
important effects on the stability and
flexibility of polypeptide chains in water
B
Because
off resonance,
◦ the polarity of peptide bond increases.
◦ the peptide bond has partial double bond
character; carbonyl carbon, Cα, and amide N are
coplanar and free rotation about the bond is
restricted.
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Which configuration is more stable?
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Used computer models of small polypeptides to
systematically vary φ and ψ with the objective of
finding stable conformations
Atoms were treated as hard spheres with
dimensions corresponding to their van der Waals
radii
For each conformation, the structure was examined
for close contacts between atoms
Therefore, φ and ψ angles which cause spheres to
collide correspond to sterically disallowed
conformations of the polypeptide backbone
The angle of the N-Cα bond to the
adjacent peptide bond is known as
phi (φ) torsion angle and the angle of
the C-Cα bond to the adjacent
peptide bond is known as psi (ψ)
Physical size of atoms or group of
atoms limits the possible ψ and φ
angles
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Plot of φ vs. ψ
The computed angles which are sterically
allowed fall on certain regions of plot
White
h
= sterically
ll disallowed
d ll
d
conformations (atoms come
closer than sum of van der
Waals radii)
Blue = sterically allowed
conformations
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φ, ψ distribution in 42 high-resolution
protein structures (x-ray crystallography)
Ramachandran Plot and Secondary
Structure
The structure of cytochrome C shows many segments
of helix and the Ramachandran plot shows a tight
grouping of φ, ψ angles near -50,-50
alpha-helix
Ramachandran plot: Cytochrome C
Ramachandran Plot
The structure of plastocyanin is composed mostly of
beta sheets; the Ramachandran plot shows values in
the –110, +130 region:
beta-sheet
¾Protein structure has a hierarchical organization.
Ramachandran plot: Plastocyanin
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Rigidity of backbone
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Interactions among amino acids
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Interactions of amino acids with water
Covalent
ƒ -Cα-C-
ƒ Disulphide
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Non-covalent
ƒ Electrostatic interaction (Salt bridge and long-range)
ƒ Hydrogen bonding
ƒ van der Waals interactions (transient, weak electrical
attraction of one atom for another)
ƒ Hydrophobic (clustering of nonpolar groups)
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Side chain of cysteine contains highly reactive thiol group; two
thiol groups form a disulfide bond
Contribute to the stability of the folded state by linking distant
parts of the polypeptide chain
Typical distance 2.2 Å and contribute ~200 KJ/mol
PDB: 5PTI
Charge-charge Interactions
Charge(The “Salt bridge” Interactions)
q1
NH3
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Charged side chains in protein can interact favorably with an
opposing charge of another side chain according to Coulomb’s law.
q2
r
O
O
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C
k q1q2
Force = ε r2
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Examples of favorable electrostatic interaction include that between
positively charged lysine and negatively charged glutamic acid.
In practice, charge-charge interactions have been shown to be
chemically significant at up to 15 Å in proteins.
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Contribute less than 5 kJmol-1 to the overall stability of a protein.
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Salts have the ability to shield electrostatic interactions.
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(Adopted from Garrett and Grisham, Biochemistry, Third Edition)
Hydrogen Bonds
N
DONOR
H
H
Hydrogen Bonds
O=C
ACCEPTOR
CC
O
δ+
δ-
δ-
H-bond length
(fixed for a given donor-acceptor pair)
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Most common type of 2˚ structural element (in globular
proteins, over 30% of the amino acids are found in helices)
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Generated by local hydrogen bonding between C=O and N-H
groups (intrachain hydrogen bonding)
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R-groups project outward, and provide the main constraints on
helical structure
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Stability is greatly enhanced by internal van der Waals contacts
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Each residue is related to the next one by a translation 1.5 Å
along the helix axis and rotation of 100 degree
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3.6 residues per turn of a helix and pitch of 5.4 Å
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Right-handed helix are energetically more favorable
Involves hydrogen bonds between backbone
groups from residues distant from each
other in the linear sequence
Antiparallel β sheet
Parallel β sheet
Two residues per turn and a translation
distance of 3.4 Å, pitch of 6.8 Å
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y The simplest secondary structure elements is the beta
turn: H-bond between the carbonyl oxygen of one
residues (n) and the amide N-H of residue n = 3, reversing
the direction of the chain
y β turn-four residues and γ turn- three residues
Hydrogen Bonds
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Hydrogen bonds between main chain NH and CO
groups
Figure
Hydrogen bonds between
i. side chains
ii. side chain - main chain
ƒ Side chains of tyrosine, threonine, and serine containing
hydroxyl group
ƒ the side chains of glutamine and asparagine with the
amide group
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Hydrogen Bonds
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These interactions occur between adjacent,
non-bonded, and uncharged atoms.
• Dipole-dipole interactions
• Dipole-induced dipole interactions
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What type of forces must be overcome within
the solid I2 when I2 dissolves in methanol,
CH3OH?
What type of forces must be disrupted between
CH3OH molecules when I2 dissolves?
What type of force exists between I2 and CH3OH
molecules in solution?
van der Waals interaction diminish rapidly as the
interacting species get farther apart; atoms that are
about 5 Å apart can have significant interactions.
A given van der waals interaction is extremely weak
4 kJmol-1 but in proteins they sum up to a
substantial energetic contribution.
• Induced dipole-induced dipole interactions (London
dispersion forces, LDF)
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Observation:
• hydrophobic groups aggregate together on the interior of the protein,
forming a hydrophobic core while hydrophilic residues are on the
outside.
• The exterior surface area of proteins can be up to 60% polar atoms
• Proteins fold to maximize their effectiveness as hydrogen-bonding
partners to water
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Water molecules surrounding the nonpolar
molecules are more ordered than water molecules
in free solution
The aggregation of nonpolar groups in water leads
to the release of water molecules
Explanation:
• When hydrophobic residues are exposed to solvent, the extended
hydrogen bonding structure of water is disrupted
• Breaking hydrogen bonds in water is energetically unfavourable
• Water molecules reorient around the hydrophobic molecule, so that
the least number of hydrogen bonds are sacrificed to accommodate it
• Burying hydrophobic residues releases water and increases entropy.
Approximate strengths of
interactions between atoms
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Interaction
Typical
distance
Energy
Salt bridge
2.8 Å
12.5 – 17 kJ/mol
Long-range
electrostatic
interactions
variable
Variable, depends
upon the
environment
Hydrogen bond
2.4 – 3.5 Å
2- 6 kJ/mol
van der Waals
interactions
3.5 – 6.0 Å
2-4 kJ/mol
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Secondary structures pack closely to one another and also intercalate
with extended polypeptide chains
Most polar residues face the outside of protein and interact with
solvent
Most hydrophobic residues face the interior of the protein and interact
with each other thereby minimizing contact with water
van der Waal’s volume is about 72-77% of the total protein volume;
about 25% is not occupied by protein atoms. These cavities provide
flexibility in protein conformation and dynamics
Random coil or loops maybe of importance in protein function
(interacting with other molecules, enzyme reactions)
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Water soluble proteins fold into compact
structure with nonpolar cores
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Membrane proteins fold into compact
structure with polar cores
charged
hydrophobic
` Folding of a globular protein is a thermodynamically
Change in Entropy and enthalpy
in protein folding
favored process, i.e. ΔG must be negative.
ΔG = ΔH - TΔS
` The folding process involves going from a multitude of
random-coil conformations to a single folded structure.
` The folding process involves a decrease in randomness
and thus a decrease in entropy -ΔS and an overall
positive contribution to ΔG. This decrease in entropy is
termed “conformational entropy”.
` An overall negative ΔG : a result of features that yield a
large negative ΔH or some other increase in entropy on
folding.
Folded Protein
Unfolded Protein
ΔH, large; ΔS, small
ΔH, small; ΔS, large
ΔG = ΔH - TΔS
ΔSsurr = −
ΔH sys
T
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ΔG, Gibbs Free Energy
Transition state,
energy barrier
ΔG
U
Unfolded
f ld d
State
Native
State
Reaction Coordinate
Anfinsen’s Experiments
Protein Folding
“The native, folded structure of a protein, under
optimal conditions, is the most energetically stable
conformation possible”
Christian Anfinsen, 1972
“Most of the information for determining the threedimensional structure of a protein is carried in its
amino acid sequence”
Anfinsen, C.B. Principles that govern the
folding of protein chains. Science 181,
223-30 (1973).
Anfinsen’s Experiments:
Unfolding of Ribonuclease
¾Ribonuclease: Involved in cleavage of nucleic acids , structure
has a combination of α and β segments, four disulfide bridges
¾Unfolding and refolding experiments were conducted
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Anfinsen’s Experiments
Reduction of Disulfide Bonds
Dialysis
β-mercaptoethanol
¾Observation:
Enzyme spontaneously folded into a catalytically active form
¾Conclusion:
The information need to specify the catalytically active structure
of ribonuclease is contained in its primary sequence
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Protein Folding is a highly Cooperative
Process
“all or none” process
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For any given protein, there is one conformation
that has significantly lower free energy than any
other state
Achieved through kinetic pathway of unstable
intermediates (not all intermediates are sampled)
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The hydrophobic effect –the clustering of
hydrophobic side chains from diverse parts of
the protein chain- causes the protein chain to
fold into compact, ordered form. Why this
effect is thermodynamically favored?
Entropy-driven process
Assisted by chaperones and protein disulfide
isomerases
Consider a protein of 100 amino acids. Assign 2
conformations to each amino acid. The total conformations of
the protein is 2100=1.27x1030. Allow 10-13 sec for the protein
to sample through one conformation in search for the overall
energy minimum. The time it needs to sample through all
conformations is
(10-13)(1.27x1030)=1.27x1017sec = 4x109 years!
Levinthal’s paradox illustrates that proteins must only sample
through limited conformations, or fold by “specific pathways”.
Much research efforts are devoted in searching for the
principles of the “specific pathways”.
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