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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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Reductive denaturation and oxidative renaturation of RNase A

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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


Determinants of Protein Folding

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


Hierarchical organization of globular proteins (subdomains)


Determinants of Protein Folding (cont.)

D. protein structures are highly adaptable

E. secondary structure can be context-dependent


“chameleon” sequence:

AWTVEKAFKTF (unfolded

free in solution)

NMR structure of protein GB1


Determinants of Protein Folding (cont.)

F. dependence of protein fold on primary sequence


created by changing 50%

of the 56 residues in GB1

not all residues have equally

important roles in specifying

a specific fold

X-ray structure of Rop protein, a homodimer of aa motifs that associate to form a 4-helix bundle


The Levinthal Paradox

2n backbone torsions, n-residue protein: ~10n

structures

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

of pathways


Experimental Methods to Monitor

Protein Folding


(UV/ VIS/ fluorescence /CD)

cold denatured proteins / T-jump

A stopped-flow device: 40 ms dead-times


molar

extinction

coefficient

UV absorbance spectra of the three aromatic amino acids, phenylalanine, tryptophan, and tyrosine


eL and eR differ (circularly

polarized light); measure ∆e

Circular dichroism (CD) spectra of polypeptides


Pulsed H/D Exchange

X-H + D2OX-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


Landscape Theory of Protein Folding

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.


Folding funnels: An idealized funnel landscape


Folding funnels: The Levinthal “golf course” landscape


Folding funnels: Classic folding landscape


Closer mimic

of an actual

folding

pathway

Folding funnels: Rugged energy surface


Folds via an ordered

pathway: involves well

defined intermediates

Polypeptide backbone and disulfide bonds of native BPTI (58 residues, three disulfide bonds)


Renaturation of BPTI: protein primary structures evolved to specify efficient folding pathways as well as stable native conformations


Folding accessory proteins

A. Protein disulfide isomerases (PDI)

B. Peptidyl prolyl cis-trans isomerases

C. Molecular chaperones


A folding accessory

protein

Reactions catalyzed by protein disulfide isomerase (PDI). (a) Reduced PDI catalyzes the rearrangement of the non-native disulfide bonds.


Reactions catalyzed by protein disulfide isomerase (PDI). (b) The oxidized PDI-dependent synthesis of disulfide bonds in proteins.


homodimer;

eukaryotic; each

subunit consists of

four domains

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.


Cys 36 is located

in a hydrophobic

patch

Oxidized PDI-a is

less stable than

reduced PDI-a

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.


Peptidyl Prolyl Cis-Trans Isomerases (PPIs)

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

Pro-containing polypeptides.

Two families: cyclophilins and FKBP12 (based on

known inhibitors)


Unfolded proteins in vivo have a great tendency to

form intramolecular and intermolecular aggregates.

Molecular chaperones prevent/reverse improper

associations, especially in multidomain and

multisubunit proteins.

Function by binding solvent-exposed hydrophobic

surfaces reversibly to promote proper folding

Many chaperones are ATPases.


Classes of Chaperones

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

in eukaryotes

D. Nucleoplasmins: acidic nuclear proteins involved in

nucleosome assembly


Chaperonins

GroEL/ES system

14 identical ~60 kD

subunits in two rings;

creates a central

cavity

Electron micrograph-derived 3D image of the Hsp60 (GroEL) chaperonin from the photosynthetic bacterium Rhodobacter sphaeroides.


X-ray structure of GroEL. (a) Side view perpendicular to the 7-fold axis.


X-ray structure of GroEL. (b) Top view along the 7-fold axis.


X-ray structure of GroES as viewed along its 7-fold axis.


cis ring

trans ring

X-ray structure of the GroEL-GroES-(ADP)7 complex.


X-ray structure of the GroEL-GroES-(ADP)7 complex.


bound ADP shown

in cis ring

X-ray structure of the GroEL-GroES-(ADP)7 complex.


apical

intermediate

equatorial

Domain movements in GroEL. (a) Ribbon diagram of a single subunit of GroEL in the X-ray structure of GroEL alone.


Domain movements in GroEL. (b) A GroEL subunit in the X-ray structure of GroEL-GroES-(ADP)7.


Domain movements in GroEL. (c) Schematic diagram indicating the conformational changes in GroEL when it binds GroES.


Apical domain of GroEL in complex with a tight-binding 12-residue polypeptide (SWMTTPWGFLHP).


(a)

(b)

Movements of the polypeptide-binding helices of GroEL.


Reaction cycle of the GroEL/ES chaperonin system in protein folding.


Models for GroEL/ES Action

A. Anfinsen cage model: folding within complex

B. Interative annealing: reversible release of partially

folded intermediates


Refolding of RiBisCO; requires

assistance to reach native state

black: no components

red: released only after

native state is reached

green: released

after one turnover

Rate of hydrogen-tritium exchange of tritiated RuBisCO.


exchange with solvent

Schematic diagram of the mechanism of stretch-induced hydrogen exchange by the GroEL/ES system.


Protein Structure Prediction

Secondary structure

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

20 residues

Pa > 1: residue occurs with greater than average frequency

in an a helix

Also applies to b-structure


H = strong former

h = former

I = weak former

i = indifferent

b = breaker

B = strong breaker

Propensities and classifications of amino acid residues for a helical and b sheet conformations.


B. Reverse turns: Rose method

Occur on the surface of a protein; locations

of minimal hydropathy (exclude helical regions)


Rationale for Observed Propensities

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


Computer-based Secondary Structure Algorithms

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

secondary structure).


Secondary structure prediction in adenylate kinase ( N-terminal 24 residues)


Tertiary Structure Prediction

a. comparative or homology modeling

b. fold recognition or threading

c. ab initio methods


Protein

Design

Structures of the second zinc finger motif of Zif268 (DNA-binding protein): X-ray structure.


sequence has

only 6 of the

28 residues

identical to Zif268 (5 are similar)

group of Phe

residues replaces

zinc finger

Structure of de novo designed peptide, FSD-1: NMR structure (a bba motif; 28 residues)


Comparison of the structures of the second zinc finger motif of Zif268 and FSD-1: best-fit superpositions of their backbones.


Protein Dynamics

Proteins undergo structural motions that have

functional significance.


Conformational fluctuations (breathing motions) in the oxygen binding protein, myoglobin.


Classes of Motions

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

displacements)

Techniques: crystallography, NMR, MD


blue = least mobile

red = most mobile

The mobility of the GroEL subunit in the X-ray structure of GroEL alone.


blue = least mobile

red = most mobile

The mobility of the GroEL subunit in the X-ray structure of the GroEL-GroES-(ADP)7 complex.


The internal motions of myoglobin as determined by a molecular dynamics simulation: the Ca backbone and the heme group.


The internal motions of myoglobin as determined by a molecular dynamics (MD) simulation: an a helix.


Detecting

infrequent

motions (time

scale of seconds)

Exchange rate of

a particular proton

correlates with the

conformational

mobility of its

surroundings

The hydrogen-tritium “exchange-out” curve for hemoglobin that has been pre-equilibrated with tritiated water.


Conformational Diseases: Amyloid and Prions

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)


Amyloid fibrils: an electron micrograph of amyloid fibrils of the protein PrP 27-30.


a

b

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.


Amyloidogenic proteins

are mutant forms of

normally occuring proteins

Lysozyme mutants

occur in familial visceral

amyloidosis

Superposition of wild-type human lysozyme and its D67H mutant.


PrionDiseases

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.


Evidence that the scrapie agent is a protein: hydroxylamine rescues diethylpyrocarbonate-inactivated scrapie reagent.


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.


Prion hypothesis: PrPSc

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)


END


Figure 9-36Molecular formula for iron-protoporphyrin IX (heme).


Figure 9-37Primary structures of some representative c-type cytochromes.


Figure 9-38Three-dimensional structures of the c-type cytochromes whose primary structures are displayed in Fig. 9-37.


Figure 9-39The two-structurally similar domains of rhodanese.


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