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Reductive denaturation and oxidative renaturation of RNase A. Plausible mechanism for the thiol- or enzyme-catalyzed disulfide interchange reaction in a protein. protein disulfide isomerase. C-chain needed to direct proper disulfide bond formation. Primary structure of porcine proinsulin.
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protein disulfide isomerase
A. helices/sheets predominate in proteins because
they fill space efficiently
B. protein folding is directed mainly by internal residues (protein
folding is driven by hydrophobic forces)
C. protein structures are organized hierarchically
D. protein structures are highly adaptable
E. secondary structure can be context-dependent
F. dependence of protein fold on primary sequence
of the 56 residues in GB1
not all residues have equally
important roles in specifying
a specific foldX-ray structure of Rop protein, a homodimer of aa motifs that associate to form a 4-helix bundle
2n backbone torsions, n-residue protein: ~10n
time to explore all structures: t = 10n/1013 s-1
for a 100-residue protein: t = 1087 s
Conclusion: proteins fold via an ordered pathway or set
coefficientUV absorbance spectra of the three aromatic amino acids, phenylalanine, tryptophan, and tyrosine
X-H + D2OX-D + HOD
Used to follow the time course of protein folding by 2D NMR
a. Denatured protein in D2O
b. Dilute with H2O and allow to fold for time tf
c. Increase pH to initiate D-H exchange (10-40 ms)
d. Lower pH; allow to completely fold
e. Determine which amide protons are protonated and deuterated
Polypeptides fold via a series of conformational
adjustments that reduce their free energy and entropy
until the native state is reached.
There is no single pathway or closely related set
of pathways that a polypeptide must follow in folding
to its native state.
The sequence information specifying a particular
fold is both distributed throughout the polypeptide
chain and highly overdetermined.
pathway: involves well
defined intermediatesPolypeptide backbone and disulfide bonds of native BPTI (58 residues, three disulfide bonds)
A. Protein disulfide isomerases (PDI)
B. Peptidyl prolyl cis-trans isomerases
C. Molecular chaperones
proteinReactions catalyzed by protein disulfide isomerase (PDI). (a) Reduced PDI catalyzes the rearrangement of the non-native disulfide bonds.
subunit consists of
Cys 36 and 39 exposed
NMR structure of the a domain of human protein disulfide isomerase (PDI-a) in its oxidized form. (a) The polypeptide backbone is shown in ribbon form.
in a hydrophobic
Oxidized PDI-a is
less stable than
NMR structure of the a domain of human protein disulfide isomerase (PDI-a) in its oxidized form. (b) The molecular structure as viewed from the bottom.
Xaa-Pro peptide bonds: ~10% cis
PPIs catalyze the otherwise slow interconversion
of Xaa-Pro peptide bonds between their cis and
trans conformations; accelerate the folding of
Two families: cyclophilins and FKBP12 (based on
form intramolecular and intermolecular aggregates.
Molecular chaperones prevent/reverse improper
associations, especially in multidomain and
Function by binding solvent-exposed hydrophobic
surfaces reversibly to promote proper folding
Many chaperones are ATPases.
A. Heat shock proteins 70: 70 kD monomeric proteins
B. Chaperonins: form large multisubunit cage-like assemblies
C. Hsp90: involved in signal transduction; very abundant
D. Nucleoplasmins: acidic nuclear proteins involved in
14 identical ~60 kD
subunits in two rings;
creates a central
cavityElectron micrograph-derived 3D image of the Hsp60 (GroEL) chaperonin from the photosynthetic bacterium Rhodobacter sphaeroides.
equatorialDomain movements in GroEL. (a) Ribbon diagram of a single subunit of GroEL in the X-ray structure of GroEL alone.
A. Anfinsen cage model: folding within complex
B. Interative annealing: reversible release of partially
assistance to reach native state
black: no components
red: released only after
native state is reached
after one turnoverRate of hydrogen-tritium exchange of tritiated RuBisCO.
a) Chou-Fasman method
Frequency at which a given aa occurs in an a helix in a set of protein structures = fa = na/n, where na = number of amino acid residues of the given type that occur in a helices, and n = total number of residues of this type in the protein set
Propensity of a particular aa residue to occur in an a helix =
Pa = fa/<fa>, where <fa> is the average value of fa for all
Pa > 1: residue occurs with greater than average frequency
in an a helix
Also applies to b-structure
h = former
I = weak former
i = indifferent
b = breaker
B = strong breakerPropensities and classifications of amino acid residues for a helical and b sheet conformations.
Occur on the surface of a protein; locations
of minimal hydropathy (exclude helical regions)
For a-helix: appears related to the amount of side-chain
hydrophobic surface buried in the protein
For Pro: low a propensity caused by strain
For Gly: low a propensity caused by reduced entropy and
lack of hydrophobic stabilization
For Ala: high a propensity caused by lack of a g substituent;
reduced entropic cost; minimal hydrophobic stabilization
Combine three or more methods: accurate to ~75%
Jpred: public domain software
The moderate accuracy is caused by failure to take tertiary
interactions into account (tertiary structure influences
a. comparative or homology modeling
b. fold recognition or threading
c. ab initio methods
DesignStructures of the second zinc finger motif of Zif268 (DNA-binding protein): X-ray structure.
only 6 of the
identical to Zif268 (5 are similar)
group of Phe
zinc fingerStructure of de novo designed peptide, FSD-1: NMR structure (a bba motif; 28 residues)
Proteins undergo structural motions that have
1. atomic fluctuations (10-15-10-11 s; 0.01 - 1Å displacements)
2. collective motions (10-12-10-3 s; 0.01 - 5 Å displacements)
3. triggered conformational changes (10-9 -103 s; 0.5 - 10 Å
Techniques: crystallography, NMR, MD
red = most mobileThe mobility of the GroEL subunit in the X-ray structure of GroEL alone.
red = most mobileThe mobility of the GroEL subunit in the X-ray structure of the GroEL-GroES-(ADP)7 complex.
scale of seconds)
Exchange rate of
a particular proton
correlates with the
mobility of its
surroundingsThe hydrogen-tritium “exchange-out” curve for hemoglobin that has been pre-equilibrated with tritiated water.
Alzheimer’s disease; transmissible spongiform encephalopathies (TSEs); amyloidoses
Common characteristic: formation of amyloid fibrils
The involved proteins assume two different
stable conformations (native and amyloid)
Fibrils consist mainly of
b-sheets whose b-strands
are perpendicular to the
fibril axis.Amyloid fibrils (PrP 27-30): Model (a) and isolated (b) b sheet.
are mutant forms of
normally occuring proteins
occur in familial visceral
amyloidosisSuperposition of wild-type human lysozyme and its D67H mutant.
Evidence that the scrapie agent is a protein: scrapie agent is inactivated by treatment with diethylpyrocarbonate, which reacts with His sidechains.
Evidence that the scrapie agent is a protein: scrapie agent is unaffected by treatment with hydroxylamine, which reacts with cytosine residues.
Prion protein conformations: NMR structure of human prion protein (PrPC). Note the disordered N-terminal tail residues (dots). PrP may be a cell-surface signal receptor.
induces the conversion of
PrPC to PrpSc
Conversion may be mediated
by a molecular chaperone.Prion protein conformations: a plausible model for the structure of PrPSc (very insoluble)