Download Lecture 10 - Columbus Labs

Class Business
Please load your paper up on Toolkit if you haven’t
already done so.
Review session on Monday at 7PM in Gilmer 190
– Q&A style so come with questions
You do not have to hand in problem set 5 and I will
post the solutions on Toolkit by tomorrow
I will handout problem set 6 at the exam
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Review of amino acids and protein
primary and secondary structure
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MCAT Question
•
Dithiothreitol (Cleland’s Reagent) will
reduce and break disulfide bonds. This
reagent will affect which amino acid?
A.
B.
C.
D.
Glutamate
Cysteine
Arginine
Selenocysteine
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MCAT Question
Enterokinase is a protease that cleaves after
lysine residues. It will sometimes cleave
at other basic residues. Which of the
following amino acids can also serve as
a target for enterokinase?
A. Proline
B. Arginine
C. Tryptophan
D. Alanine
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Name two secondary structural elements
found in proteins?
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Ramachandran Plot
α-helix
phi = -60 degrees
psi = -45 degrees
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Tertiary Structure
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Hydrophobic Interactions
The tertiary structures of water-soluble proteins have
features in common:
(1) an interior formed of amino acids with hydrophobic
side chains and
(2) a surface formed largely of hydrophilic amino acids
that interact with the aqueous environment
(3) Atoms are packed very close; however, cavities do
occur and maybe functionally important
Cross-sectional view
Hydrophobic residues
Charged residues
Other residues in white
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Myoglobin
Hydrophobic effect
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Many α helices and β strands are amphipathic; that is, the α helix or β
strand has a hydrophobic face, which points into the protein interior, and
a more polar face, which points into solution.
An amphiphilic helix
in flavodoxin:
A nonpolar helix in
citrate synthase:
A polar helix in
calmodulin:
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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
• Simplest way to pack
helices - short
connecting loops and
antiparallel packing
• The helix bundle
often involves a slight
(15 degree) lefthanded twist
• The globin proteins myoglobin and
hemoglobin - are
antiparallel alpha
proteins
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Coiled-coil motif – parallel or antipallel
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Parallel or Mixed Beta Sheet Proteins
• 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
Beta-barrels
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Parallel Beta-sheet Proteins
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Antiparallel Beta Sheets
• 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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Greek Key Antiparallel Beta-sheets
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More Antiparallel Beta Sheets
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MCAT Question
Parallel and non-parallel beta-pleated
sheets are stabilized by which of the
following interactions?
A. Covalent bonds
B. Electrostatic interactions
C. Hydrogen bonds
D. Hydrophilic interactions
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Metal-Rich and Disulfide-rich Proteins
•
Conformations usually heavily influenced by metals and/or disulfide
bridges
• These proteins can be unstable if the metals are removed or the
disulfides are reduced: “structurally essential”
• Some metals are involved in activity
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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
•
•
Found in hair, fingernails, claws, horns and beaks
Sequence consists of 311-314 residue alpha helical rod
segments capped with non-helical N- and C-termini
• Primary structure of helical rods consists of 7-residue repeats: (ab-c-d-e-f-g)n, where a and d are nonpolar. Promotes association
of helices!
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Beta
Keratin
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
– Class I – heterotrimer (bones, tendons, and skin)
– Class II (cartilage) and III – homotrimers (blood
vessels
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Collagen
• 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.
Posttranslational modifications
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The Collagen Triple Helix
• 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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MCAT Question
The collagen triple helix interior requires an
amino acid with a small side chain.
Which of the following would most likely
be found in the interior of a collagen
molecule?
A. Methionine
B. Aspartate
C. Tryptophan
D. Glycine
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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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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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Protein Modules
•
•
•
•
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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Protein Folding
Cyrus Levinthal calculated that, if each residue can assume three different
conformations, the total number of structures would be
3100, which is equal to 5 × 1047.
If it takes 10-13 s to convert one structure into another,
the total search time would be 5 × 1047 × 10-13 s, which is equal to 5 × 1034 s, or
1.6 × 1027 years.
The enormous difference between calculated and actual folding times is called
Levinthal's paradox.
Richard Dawkins, in The Blind Watchmaker, asked how long it would take a monkey
poking randomly at a typewriter to reproduce Hamlet's remark to Polonius, “Methinks
it is like a weasel”.
≈ 1040
However, suppose that we preserved each correct character and allowed the
monkey to retype only the wrong ones.
only a few thousand keystrokes, on average, would be needed.
The crucial difference between these cases is that the first employs a completely
random search, whereas, in the second, partly correct intermediates are retained.
The essence of protein folding is the retention of partly correct intermediates.
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Protein Folding
Energy Landscape
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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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What are the structural and functional
advantages driving quaternary association?
• Stability: reduction of surface to volume ratio
• Bringing catalytic sites together
• Cooperativity
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Symmetry in Quaternary Structure
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Quaternary Structures of Hemerythrin
Monomer
T. zostericola
Octamer
P. gouldii
Trimer
Siphonosoma
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