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

Primary structure of porcine proinsulin

C-chain neededto direct properdisulfide bond

formation

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 adaptableE. secondary structure can be context-dependent

NMR structure of protein GB1

“chameleon” sequence:AWTVEKAFKTF (unfolded

free in solution)

Determinants of Protein Folding (cont.)

F. dependence of protein fold on primary sequence

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

created by changing 50%of the 56 residues in GB1

not all residues have equallyimportant roles in specifying

a specific fold

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 setof pathways

Experimental Methods to MonitorProtein Folding

A stopped-flow device: 40 s dead-times

(UV/ VIS/ fluorescence /CD)

cold denatured proteins / T-jump

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

molarextinctioncoefficient

Circular dichroism (CD) spectra of polypeptides

L and R differ (circularlypolarized light); measure ∆

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 D2Ob. 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 folde. Determine which amide protons are protonated and deuterated

Landscape Theory of Protein Folding

Polypeptides fold via a series of conformationaladjustments that reduce their free energy and entropy

until the native state is reached.

There is no single pathway or closely related setof pathways that a polypeptide must follow in folding

to its native state.

The sequence information specifying a particularfold 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

Folding funnels: Rugged energy surface

Closer mimicof an actual

foldingpathway

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

Folds via an orderedpathway: involves welldefined intermediates

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 isomerasesC. Molecular chaperones

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

A folding accessoryprotein

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

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.

homodimer;eukaryotic; each

subunit consists offour domains

Cys 36 and 39 exposed

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.

Cys 36 is locatedin a hydrophobic

patch

Oxidized PDI-a isless stable thanreduced PDI-a

Peptidyl Prolyl Cis-Trans Isomerases (PPIs)

Xaa-Pro peptide bonds: ~10% cis

PPIs catalyze the otherwise slow interconversionof Xaa-Pro peptide bonds between their cis andtrans conformations; accelerate the folding ofPro-containing polypeptides.

Two families: cyclophilins and FKBP12 (based onknown inhibitors)

Unfolded proteins in vivo have a great tendency toform intramolecular and intermolecular aggregates.

Molecular chaperones prevent/reverse improperassociations, 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 abundantin eukaryotes

D. Nucleoplasmins: acidic nuclear proteins involved innucleosome assembly

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

Chaperonins

GroEL/ES system

14 identical ~60 kDsubunits in two rings;

creates a centralcavity

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.

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

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

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

apical

intermediate

equatorial

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).

Movements of the polypeptide-binding helices of GroEL.

(a) (b)

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 partiallyfolded intermediates

Rate of hydrogen-tritium exchange of tritiated RuBisCO.

Refolding of RiBisCO; requiresassistance to reach native state

black: no components

red: released only afternative state is reached

green: releasedafter one turnover

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

exchange with solvent

Protein Structure Prediction

Secondary structurea) Chou-Fasman method

Frequency at which a given aa occurs in an helix in a set of protein structures = f = n/n, where n = number of amino acid residues of the given type that occur in helices, and n = total number of residues of this type in the protein set

Propensity of a particular aa residue to occur in an helix =P = f/<f>, where <f> is the average value of f for all20 residues

P > 1: residue occurs with greater than average frequencyin an helix

Also applies to -structure

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

H = strong formerh = formerI = weak formeri = indifferentb = breakerB = strong breaker

B. Reverse turns: Rose method

Occur on the surface of a protein; locationsof minimal hydropathy (exclude helical regions)

Rationale for Observed Propensities

For -helix: appears related to the amount of side-chainhydrophobic surface buried in the protein

For Pro: low propensity caused by strain

For Gly: low propensity caused by reduced entropy andlack of hydrophobic stabilization

For Ala: high propensity caused by lack of a 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 tertiaryinteractions into account (tertiary structure influencessecondary structure).

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

Tertiary Structure Prediction

a. comparative or homology modelingb. fold recognition or threadingc. ab initio methods

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

ProteinDesign

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

sequence hasonly 6 of the28 residues

identical to Zif268 (5 are similar)

group of Pheresidues replaces

zinc finger

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

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

blue = least mobilered = most mobile

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

blue = least mobilered = most mobile

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

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

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

Detectinginfrequent

motions (timescale of seconds)

Exchange rate ofa particular protoncorrelates with the

conformational mobility of itssurroundings

Conformational Diseases: Amyloid and Prions

Alzheimer’s disease; transmissible spongiform encephalopathies (TSEs); amyloidoses

Common characteristic: formation of amyloid fibrils

The involved proteins assume two differentstable conformations (native and amyloid)

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

Amyloid fibrils (PrP 27-30): Model (a) and isolated (b) sheet.

a b

Fibrils consist mainly of-sheets whose -strandsare perpendicular to the

fibril axis.

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

Lysozyme mutants

occur in familial visceral

amyloidosis

Amyloidogenic proteinsare mutant forms of

normally occuring proteins

Evidence that the scrapie agent is a protein: scrapie agent is inactivated by treatment with diethylpyrocarbonate, which reacts with His sidechains.

Prion Diseases

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 protein conformations: a plausible model for the structure of PrPSc (very insoluble)

Prion hypothesis: PrPSc induces the conversion of

PrPC to PrpSc

Conversion may be mediatedby a molecular chaperone.

END

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

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

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

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