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Biochemistry 2/e - Garrett & Grisham
Proteins: Their Structure and
Biological Functions
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Biological Functions of
Proteins
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Proteins are the agents of biological function
Enzymes - Ribonuclease
Regulatory proteins - Insulin, PCNA
Transport proteins - Hemoglobin
Structural proteins - Collagen
Contractile proteins - Actin, Myosin
Protective proteins - Antifreeze proteins
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Biochemistry 2/e - Garrett & Grisham
Protein structure often
provides clues about protein
function
Unrelated proteins assume
similar structures to fulfill
common functions
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Biochemistry 2/e - Garrett & Grisham
Proteins are Linear Polymers of Amino Acids
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Biochemistry 2/e - Garrett & Grisham
Peptides
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Short polymers of amino acids
Each unit is called a residue
2 residues - dipeptide
3 residues - tripeptide
12-20 residues - oligopeptide
many - polypeptide
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Biochemistry 2/e - Garrett & Grisham
Protein
One or more polypeptide chains
• One polypeptide chain - a monomeric protein
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More than one - multimeric protein
Homomultimer - one kind of chain
Heteromultimer - two or more different chains
Hemoglobin, for example, is a heterotetramer;
it has two alpha chains and two beta chains
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Biochemistry 2/e - Garrett & Grisham
Proteins - Large and Small
• Insulin - A chain of 21 residues, B chain of
30 residues -total mol. wt. of 5,733
• Glutamine synthetase - 12 subunits of 468
residues each - total mol. wt. of 600,000
• Connectin proteins - alpha - MW 2.8 million!
• beta connectin - MW of 2.1 million, with a
length of 1000 nm -it can stretch to 3000
nm!
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Biochemistry 2/e - Garrett & Grisham
Amino acid composition provides some (limited)
clues about protein structure-function
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Biochemistry 2/e - Garrett & Grisham
The Sequence of Amino Acids
in a Protein
• is a unique characteristic of every
protein
• is encoded by the nucleotide sequence
of DNA
• is thus a form of genetic information
• is read from the amino terminus to the
carboxyl terminus
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Biochemistry 2/e - Garrett & Grisham
• The levels of protein structure
- Primary
sequence
- Secondary local structures
- Tertiary
overall 3-dimensional
shape
- Quaternary subunit organization
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Biochemistry 2/e - Garrett & Grisham
What forces determine the
structure?
• Primary structure - determined by
covalent bonds
• Secondary, Tertiary, Quaternary
structures - all determined by weak
forces
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Biochemistry 2/e - Garrett & Grisham
The Role of the Sequence in
Protein Structure
All of the information necessary for
folding the peptide chain into its "native”
structure is contained in the primary
amino acid structure of the peptide.
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Biochemistry 2/e - Garrett & Grisham
The sequence of ribonuclease A
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Biochemistry 2/e - Garrett & Grisham
Sequence Determination
Frederick Sanger was the first - in 1953, he
sequenced the two chains of insulin.
• Sanger's results established that all of the
molecules of a given protein have the same
sequence
• Proteins can be sequenced in two ways:
- real amino acid sequencing
- sequencing the corresponding DNA in
the gene
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Biochemistry 2/e - Garrett & Grisham
Nature of Protein Sequences
• Sequences and composition reflect the
function of the protein:
• Membrane proteins have stretches of
hydrophobic residues, whereas fibrous
proteins may have atypical sequences
• Homologous proteins from different
organisms have similar sequences e.g.,
cytochrome c is highly conserved
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Phylogeny of Cytochrome c
• The number of amino acid differences
between two cytochrome c sequences
is proportional to the phylogenetic
difference between the species from
which they are derived
• This observation can be used to build
phylogenetic trees of proteins
• This is the basis for studies of molecular
evolution
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
So, how do proteins fold?
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Biochemistry 2/e - Garrett & Grisham
Proteins are Linear Polymers of Amino Acids
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
The Coplanar Nature of the Peptide Bond
Six atoms of the peptide group lie in a plane
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Biochemistry 2/e - Garrett & Grisham
Configuration and
conformation are
not the same
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Biochemistry 2/e - Garrett & Grisham
The Peptide Bond
• is usually found in the trans conformation
• has partial (40%) double bond character
• is about 0.133 nm long - shorter than a typical
single bond but longer than a double bond
• Due to the double bond character, the six atoms
of the peptide bond group are always planar.
• N partially positive; O partially negative
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Biochemistry 2/e - Garrett & Grisham
Secondary Structure
The atoms of the peptide bond lie in a plane
• The resonance stabilization energy of the planar
structure is 88 kJ/mol
• A twist about the C-N bond involves a twist energy
of 88 kJ/mol times the square of the twist angle.
• Twists can occur about either of the bonds linking
the alpha carbon to the other atoms of the peptide
backbone
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Biochemistry 2/e - Garrett & Grisham
Consequences of the Amide Plane
Two degrees of freedom per residue for the peptide chain
• Angle about the C(alpha)-N bond is denoted phi
• Angle about the C(alpha)-C bond is denoted psi
• The entire path of the peptide backbone is known if all
phi and psi angles are specified
• Some values of phi and psi are more likely than others.
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Steric Constraints on phi & psi
Unfavorable overlap precludes some
combinations of phi and psi
• phi = 0, psi = 180 is unfavorable
• phi = 180, psi = 0 is unfavorable
• phi = 0, psi = 0 is unfavorable
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Classes of Secondary Structure
All these are local structures that are
stabilized by hydrogen bonds
• Alpha helix
• Beta sheet (composed of "beta strands")
• Tight turns (aka beta turns or beta bends)
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Biochemistry 2/e - Garrett & Grisham
The Alpha Helix
• First proposed by Linus Pauling and
Robert Corey in 1951
• A ubiquitous component of proteins
• Stabilized by H-bonds
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
The Alpha Helix
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Residues per turn: 3.6
Rise per residue: 1.5 Angstroms
Rise per turn (pitch): 3.6 x 1.5A = 5.4 Angstroms
The backbone loop that is closed by any H-bond
in an alpha helix contains 13 atoms
• phi = -60 degrees, psi = -45 degrees
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
The Beta-Pleated Sheet
Composed of beta strands
• Also first postulated by Pauling and Corey,
1951
• Strands may be parallel or antiparallel
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
The Beta Turn
(aka beta bend, tight turn)
• allows the peptide chain to reverse
direction
• carbonyl C of one residue is H-bonded
to the amide proton of a residue three
residues away
• proline and glycine are prevalent in beta
turns
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Steric Constraints on phi & psi
• G. N. Ramachandran was the first to
demonstrate the convenience of plotting
phi,psi combinations from known protein
structures
• The sterically favorable combinations
are the basis for preferred secondary
structures
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Predictive Algorithms
If the sequence holds the secrets of folding, can we
figure it out?
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Biochemistry 2/e - Garrett & Grisham
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Biochemistry 2/e - Garrett & Grisham
Tertiary Structure
Several important principles:
• The backbone links between elements
of secondary structure are usually short
and direct
• Proteins fold to make the most stable
structures (make H-bonds and minimize
solvent contact
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Biochemistry 2/e - Garrett & Grisham
Tertiary Structure
So, how do proteins fold?
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Biochemistry 2/e - Garrett & Grisham
Weak Forces are Responsible
for Protein Folding
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What are they?
What are the relevant numbers?
van der Waals: 0.4 - 4 kJ/mol
hydrogen bonds: 12-30 kJ/mol
ionic bonds: 20 kJ/mol
hydrophobic interactions: <40 kJ/mol
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Biochemistry 2/e - Garrett & Grisham
Thermodynamics of Folding
• Separate the enthalpy and entropy terms for the
peptide chain and the solvent
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Biochemistry 2/e - Garrett & Grisham
The largest favorable contribution to folding is the entropy term
for the interaction of nonpolar residues with the solvent
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Biochemistry 2/e - Garrett & Grisham
Tertiary Structure
Several important principles:
• Secondary structures form wherever
possible (due to formation of large
numbers of H-bonds)
• Helices and sheets often pack close
together
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
How do proteins recognize and
interpret the folding information?
• Certain loci along the chain may act as
nucleation points
• Protein chain must avoid local energy
minima
• Chaperones may help
• Peptide chains, composed of L-amino
acids, have a tendency to undergo a
"right-handed twist"
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Globular Proteins
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Some design principles
Most polar residues face the outside of the
protein and interact with solvent
Most hydrophobic residues face the interior of
the protein and interact with each other
Packing of residues is close
However, ratio of vdw volume to total volume is
only 0.72 to 0.77, so empty space exists
The empty space is in the form of small cavities
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Globular Proteins
The Forces That Drive Folding
• Peptide chain must satisfy the constraints
inherent in its own structure
• Peptide chain must fold so as to "bury" the
hydrophobic side chains, minimizing their
contact with water
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Biochemistry 2/e - Garrett & Grisham
Globular Proteins
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More design principles
"Random coil" is not random
Structures of globular proteins are not
static
Various elements of protein move to
different degrees
Some segments of proteins are very
flexible and disordered
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Biochemistry 2/e - Garrett & Grisham
An amphiphilic helix
in flavodoxin:
A nonpolar helix in
citrate synthase:
A polar helix in
calmodulin:
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Biochemistry 2/e - Garrett & Grisham
Protein Modules
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An important insight into protein structure
Many proteins are constructed as a composite of
two or more "modules" or domains
Each of these is a recognizable domain that can
also be found in other proteins
Sometimes modules are used repeatedly in the
same protein
There is a genetic basis for the use of modules in
nature
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Molecular Chaperones
• Why are chaperones needed if the
information for folding is inherent in the
sequence?
– to protect nascent proteins from the
concentrated protein matrix in the cell and
perhaps to accelerate slow steps
• Chaperone proteins were first identified
as "heat-shock proteins" (hsp60 and
hsp70)
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Other Chemical Groups in
Proteins
Proteins may be "conjugated" with other
chemical groups
• If the non-amino acid part of the protein is
important to its function, it is called a
prosthetic group.
• Be familiar with the terms: glycoprotein,
lipoprotein, nucleoprotein, phosphoprotein,
metalloprotein, hemoprotein, flavoprotein.
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham
Quaternary Structure
What are the forces driving quaternary association?
• Typical Kd for two subunits: 10-8 to 10-16M!
• These values correspond to energies of 50-100
kJ/mol at 37 C
• Entropy loss due to association - unfavorable
• Entropy gain due to burying of hydrophobic
groups - very favorable!
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Biochemistry 2/e - Garrett & Grisham
What are the structural and functional
advantages driving quaternary association?
Know these!
• Stability: reduction of surface to volume
ratio
• Genetic economy and efficiency
• Bringing catalytic sites together
• Cooperativity
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Biochemistry 2/e - Garrett & Grisham
Copyright © 1999 by Harcourt Brace & Company