Chapter 6 Slides

Report
Biochemistry 2/e - Garrett & Grisham
Chapter 6
Proteins: Secondary, Tertiary, and
Quaternary Structure
to accompany
Biochemistry, 2/e
by
Reginald Garrett and Charles Grisham
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Biochemistry 2/e - Garrett & Grisham
Outline
• 6.1 Forces Influencing Protein Structure
• 6.2 Role of the Amino Acid Sequence in
Protein Structure
• 6.3 Secondary Structure of Proteins
• 6.4 Protein Folding and Tertiary Structure
• 6.5 Subunit Interactions and Quaternary
Structure
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Biochemistry 2/e - Garrett & Grisham
6.1 The Weak Forces
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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
6.2 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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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
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6.3 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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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.
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Biochemistry 2/e - Garrett & Grisham
The angles phi and
psi are shown here
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Steric Constraints on phi & psi
Unfavorable orbital 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
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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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Classes of Secondary Structure
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•
•
•
•
All these are local structures that are
stabilized by hydrogen bonds
Alpha helix
Other helices
Beta sheet (composed of "beta strands")
Tight turns (aka beta turns or beta bends)
Beta bulge
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The Alpha Helix
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•
•
•
Read the box on page 167
First proposed by Linus Pauling and
Robert Corey in 1951
Identified in keratin by Max Perutz
A ubiquitous component of proteins
Stabilized by H-bonds
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Biochemistry 2/e - Garrett & Grisham
The Alpha Helix
Know these numbers
•
•
•
•
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
• The non-integral number of residues per turn
was a surprise to crystallographers
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The Beta-Pleated Sheet
Composed of beta strands
• Also first postulated by Pauling and Corey,
1951
• Strands may be parallel or antiparallel
• Rise per residue:
•
– 3.47 Angstroms for antiparallel strands
– 3.25 Angstroms for parallel strands
– Each strand of a beta sheet may be pictured
as a helix with two residues per turn
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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
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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
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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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Fibrous Proteins
• Much or most of the polypeptide chain
is organized approximately parallel to a
single axis
• Fibrous proteins are often mechanically
strong
• Fibrous proteins are usually insoluble
• Usually play a structural role in nature
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Alpha Keratin
Read the box on page 175
• Found in hair, fingernails, claws, horns and
beaks
• Sequence consists of 311-314 residue alpha
helical rod segments capped with non-helical Nand C-termini
• Primary structure of helical rods consists of 7residue repeats: (a-b-c-d-e-f-g)n, where a and d
are nonpolar. Promotes association of helices!
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Beta Keratin
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•
•
•
Proteins that form extensive beta sheets
Found in silk fibers
Alternating sequence:
Gly-Ala/Ser-Gly-Ala/Ser....
Since residues of a beta sheet extend
alternately above and below the plane of the
sheet, this places all glycines on one side and
all alanines and serines on other side!
This allows Glys on one sheet to mesh with
Glys on an adjacent sheet (same for Ala/Sers)
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Collagen - A Triple Helix
Principal component of connective tissue
(tendons, cartilage, bones, teeth)
• basic unit is tropocollagen:
– three intertwined polypeptide chains (1000
residues each
– MW = 285,000
– 300 nm long, 1.4 nm diameter
– unique amino acid composition
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Collagen
The secrets of its a.a. composition...
• Nearly one residue out of three is Gly
• Proline content is unusually high
• Unusual amino acids found:
– 4-hydroxyproline
– 3-hydroxyproline
– 5-hydroxylysine
– Pro and HyPro together make 30% of res.
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The Collagen Triple Helix
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•
•
•
•
A case of structure following composition
The unusual amino acid composition of
collagen is unsuited for alpha helices OR beta
sheets
But it is ideally suited for the collagen triple
helix: three intertwined helical strands
Much more extended than alpha helix, with a
rise per residue of 2.9 Angstroms
3.3 residues per turn
Long stretches of Gly-Pro-Pro/HyP
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Collagen Fibers
Staggered arrays of tropocollagens
• Banding pattern in EMs with 68 nm repeat
• Since tropocollagens are 300 nm long,
there must be 40 nm gaps between
adjacent tropocollagens (5x68 = 340
Angstroms)
• 40 nm gaps are called "hole regions" - they
contain carbohydrate and are thought to
be nucleation sites for bone formation
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Structural basis of the
collagen triple helix
• Every third residue faces the crowded center
of the helix - only Gly fits here
• Pro and HyP suit the constraints of phi and psi
• Interchain H-bonds involving HyP stabilize
helix
• Fibrils are further strengthened by intrachain
lysine-lysine and interchain hydroxypyridinium
crosslinks
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Globular Proteins
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•
•
•
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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
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An amphiphilic helix
in flavodoxin:
A nonpolar helix in
citrate synthase:
A polar helix in
calmodulin:
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Globular Proteins
•
•
•
•
•
More design principles
"Random coil" is not random
Structures of globular proteins are not
static
Various elements and domains of protein
move to different degrees
Some segments of proteins are very
flexible and disordered
Know the kinds and rates of protein motion
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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
• Peptide chains, composed of L-amino
acids, have a tendency to undergo a
"right-handed twist"
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A New Way to Look at
Globular Proteins
Look for "layer structures"
• Helices and sheets often pack in layers
• Hydrophobic residues are sandwiched
between the layers
• Outside layers are covered with mostly
polar residues that interact favorably
with solvent
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Classes of Globular Proteins
•
•
•
•
Jane Richardson's classification
Antiparallel alpha helix proteins
Parallel or mixed beta sheet proteins
Antiparallel beta sheet proteins
Metal- and disulfide-rich proteins
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Antiparallel Alpha Helical
Proteins
See Figure 6.29 for some examples
• Simplest way to pack helices - short
connecting loops and antiparallel packing
• The helix bundle often involves a slight (15
degree) left-handed twist
• The globin proteins - myoglobin and
hemoglobin - are antiparallel alpha
proteins
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Parallel or Mixed Beta Sheet
Proteins
See Figure 6.30, 6.31
• Parallel beta sheets distribute nonpolar
residues on both sides of the beta sheet
• This means that both faces of the sheet must
be protected from solvent
• Thus parallel beta sheets are core structures
• Parallel beta barrels are in this class
• Doubly wound parallel beta sheets also
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Antiparallel Beta Sheets
See Figures 6.32, 6.33, 6.34
• Antiparallel beta sheets place nonpolar
residues on only one face of the sheet
• Only one face must be protected from solvent
• Thus antiparallel beta sheet proteins may
contain as few as two layers
• Possibilities: barrels, beta sandwiches and
sheets covered by helices on one face only
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Metal-Rich and Disulfide-rich
Proteins
See Figure 6.35
• Usually less than 100 residues
• Conformations usually heavily influenced
by metals and/or disulfide bridges
• These proteins are usually unstable if the
metals are removed or the disulfides are
reduced
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Thermodynamics of Folding
Read the box on page 192
• Separate the enthalpy and entropy terms for
the peptide chain and the solvent
• Further distinguish polar and nonpolar
groups
• The largest favorable contribution to folding is
the entropy term for the interaction of
nonpolar residues with the solvent
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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)
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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
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Predictive Algorithms
If the sequence holds the secrets of folding, can
we figure it out?
• Many protein chemists have tried to predict
structure based on sequence
– Chou-Fasman: each amino acid is assigned a
"propensity" for forming helices or sheets
– Chou-Fasman is only modestly successful and
doesn't predict how sheets and helices arrange
– George Rose may be much closer to solving the
problem. See Proteins 22, 81-99 (1995)
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Modeling protein
folding with Linus
(George Rose)
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Ken Dill’s folding
funnel.
Unfolded structures lie
around the top. As the
protein folds, it falls
down the wall of the
energy funnel to more
stable conformations.
The native, folded
structure is at the
bottom.
Nature Structural Biol.
4, 10-19 (1997).
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6.5 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
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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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