Guanidine hydrochloride induced unfolding of yeast iso-2 cytochrome c

Nall, Barry T.; Landers, Terry A.

doi:10.1021/bi00522a008

Cited by 70 publications

(87 citation statements)

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“…However, throughout the entire unfolding branch of the T~ kinetic phase, unfolding of N52I iso-2 is much slower than unfolding of iso-2. The iso-2 kinetic data presented in Figures 6 and S1 (supplementary material) are in good agreement with previous results (Nall & Landers, 1981;Nall, 1983;Osterhout & Nall, 1985;Nall et al, 1988). The rate of slow phase (T~,,) fluorescence-detected aThe fluorescence equilibrium unfolding parameters for iso-2 and N52I iso-2, at 20°C, 0.1 M sodium phosphate, pH 6.0, and protein concentrations of 4.1 pM.…”

Section: Folding/unjolding Kineticssupporting

confidence: 88%

“…One test for structural and energetic similarity between the folding transition state and the native conformation is the relationship between equilibrium free energies and activation free energies of folding (Matouschek & Fersht, 1993;Matthews & Fersht, 1995). Tests for a relationship between stability and folding rates for cytochromes c have compared the kinetics of folding of yeast is0 cytochromes c (Nall & Landers, 1981;Mines et al, 1996) to the folding kinetics of the more stable horse cytochrome c (Ikai et al, 1973;Tsong, 1976).…”

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“…There are two ways of testing the linear free energy hypothesis: refolding rates for cytochrome c homologues can be compared (1) at the same thermodynamic stability by adjusting the final denaturant concentration, or (2) at different t h e m e dynamic stabilities under the same refolding conditions, for example, same denaturant concentration, temperature, pH. The observation that folding rates for horse and yeast iso-2 cytochromes c are similar when compared at the same thermodynamic stability (Nall & Landers, 1981) is in accord with the linear free-energy hypothesis. Refolding compared at different thermodynamic stabilities is also in accord with a linear relationship between equilibrium free energies and activation free energies for folding: folding of the more stable horse cytochrome c is substantially faster than folding of less stable yeast iso-1 (Mines et al, 1996).…”

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Refolding rate of stability‐enhanced cytochrome c is independent of thermodynamic driving force

McGee

Nall

1998

Protein Science

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N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 3096, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.Keywords: free energy; global suppressors; iso-2 cytochrome c; protein folding; yeastThe stability enhancing properties of the asparagine to isoleucine (N52I) substitution at position 52 in cytochrome c are well known from extensive thermodynamic and X-ray crystallographic studies of N52I iso-1 cytochrome c (Das et al., 1989;Hickey et al., 1991;Berghuis et al., 1994;Komar-Panicucci et al., 1994) and N52I iso-2 cytochrome c (Fig. 1) (McGee et al., 1996). For example, scanning calorimetry measurements of thermal unfolding of N52I iso-2 show that the N52I substitution increases the stability of both the oxidized and reduced forms by 2-3 kcal/mol (McGee et al., 1996). The N52I substitution is also known to act as a "global suppressor" mutation (Shortle & Lin, 1985) by reverting other nonfunctional (second site) mutations to yield functional cytochrome c (Berroteran & Hampsey, 1991). The X-ray crystallographic structures of N52I iso-1 (Berghuis et al., 1994) and N52I iso-2 (McGee et al., 1996) show a substantial ...

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Section: Folding/unjolding Kineticssupporting

confidence: 88%

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Refolding rate of stability‐enhanced cytochrome c is independent of thermodynamic driving force

McGee

Nall

1998

Protein Science

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“…The control strain, B-7533, has the same genotype, except the wild-type CYCl + gene is present. Protein was purified by a variation on methods described previously (Sherman et al, 1968;Nall & Landers, 1981).…”

Section: Yeast Strains Mutagenesis and Protein Purificationmentioning

confidence: 99%

The structure and function of omega loop A replacements in cytochrome c

et al. 1993

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The structural and functional consequences of replacing Q-loop A (residues 18-32) in yeast iso-1-cytochrome c with the corresponding loop of Rhodospirillum rubrum cytochrome c2 have been examined. The three-dimensional structure of this loop replacement mutant RepA2 cytochrome e, and a second mutant RepA2(Val20) cytochrome c in which residue 20 was back substituted to valine, were determined using X-ray diffraction techniques. A change in the molecular packing is evident in the RepA2 mutant protein, which has a phenylalanine at position 20, a residue considerably larger than the valine found in wild-type yeast iso-I-cytochrome e. The side chain of Phe 20 is redirected toward the molecular surface, altering the packing of this region of 0-loop A with the hydrophobic core of the protein. In the RepA2(Val20) structure, Q-loop A contains a valine at position 20, which restores the original wild-type packing arrangement of the hydrophobic core. Also, as a result of Q-loop A replacement, residue 26 is changed from a histidine to asparagine, which results in displacements of the main-chain atoms near residue 44 to which residue 26 is hydrogen bonded. In vivo studies of the growth rate of the mutant strains on nonfermentable media indicate that the RepA2(Val20) cytochrome c behaves much like the wild-type yeast iso-1 protein, whereas the stability and function of the RepA2 cytochrome c showed a temperature dependence. The midpoint reduction potential measured by cyclic voltammetry of the RepA2 mutant is 271 mV at 25 "C. This is 19 mV less than the wildtype and RepA2(Val20) proteins (290 mV) and may result from disruption of the hydrophobic packing in the heme pocket and increased mobility of Q-loop A in RepA2 cytochrome c. The temperature dependence of the reduction potential is also greatly enhanced in the RepA2 protein.

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“…Mitochondrial c-type cytochromes are among the best characterized proteins. Several species have been studied by X-ray crystallography [12][13][14] , and extensive protein-folding studies have been reported [15][16][17][18][19][20][21] . …”

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Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR

Röder

Elöve

Englander

1988

Nature

889

749

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To understand the process of protein folding, it will be necessary to obtain detailed structural information on folding intermediates. This difficult problem is being studied by using hydrogen exchange and rapid mixing to label transient structural intermediates, with subsequent analysis of the proton-labelling pattern by two-dimensional nuclear magnetic resonance spectroscopy. Results for cytochrome c show that the method provides the spatial and temporal resolution necessary to monitor structure formation at many defined sites along the polypeptide chain on a timescale ranging from milliseconds to minutes.A MAJOR obstacle in the ongoing effort to discover the principles of protein folding is the elusive nature of structural intermediates on the folding pathway. The folding transition for globular proteins is often highly cooperative, which makes it difficult to study folding intermediates at equilibrium. Evidence for kinetic intermediates has been found for several proteins 1 , but the structural description of these transient states is a formidable experimental challenge. One approach relies on disulphide reactions to trap folding intermediates in stable form (reviewed in ref.2). We chose a different method which makes use of hydrogendeuterium exchange to label NH sites on the polypeptide chain that are still unprotected at defined time points during refolding, followed by two-dimensional nuclear magnetic resonance (2-D NMR) analysis of the refolded protein to determine the amount of proton label trapped at points throughout the structure. One can thus follow the time course of structure formation for those regions of the protein where labile hydrogens become protected against exchange on refolding.The application of hydrogen exchange labelling methods for kinetic folding studies was pioneered by Baldwin and coworkers 3,4 , who initially used tritium exchange and manual mixing to study the slow folding phase of ribonuclease A. The power of this approach is significantly enhanced by the use of hydrogen-deuterium exchange in conjunction with rapid mixing methods, and the subsequent proton-resolved analysis of the labels trapped during refolding using proton NMR (ref. 5). The feasibility of this method was demonstrated by Roder and Wüthrich 6 in a study of early structural events in the refolding of bovine pancreatic trypsin inhibitor using one-dimensional NMR. The development of 2-D NMR methods 7-9 now makes it possible to monitor a comprehensive set of individual protons at known locations throughout the protein structure. Hydrogen exchange adds the ability to resolve structural events on a millisecond timescale to the inherent spatial resolution of 2-D NMR.The hydrogen exchange labelling results presented here provide an initial picture of the folding process for cytochrome c in terms of the time-resolved formation of hydrogenbonded structure. Distinct kinetic patterns are observed for amide protons located in different parts of the protein. Several amide sites on the two terminal helices acquire over...

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Guanidine hydrochloride induced unfolding of yeast iso-2 cytochrome c

Cited by 70 publications

References 38 publications

Refolding rate of stability‐enhanced cytochrome c is independent of thermodynamic driving force

Refolding rate of stability‐enhanced cytochrome c is independent of thermodynamic driving force

The structure and function of omega loop A replacements in cytochrome c

Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR

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