Test of the extended two-state model for the kinetic intermediates observed in the folding transition of ribonuclease A

Nall, Barry T.; Garel, Jean‐Renaud; Baldwin, Robert L.

doi:10.1016/0022-2836(78)90231-0

Cited by 151 publications

(126 citation statements)

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“…3). This difference in the denaturant dependence between the unfolding and the refolding reaction was previously observed for several proteins like RNase A (Nall et al, 1978;Kiefhaber & Schmid, 1991), RNase T1 , and barnase (Matouschek et al, 1990).…”

Section: Properties Of the Folding Reactionsupporting

confidence: 69%

“…Already at low GdmCl concentrations the native state is at the edge of its stability, and any partially folded structures, which are always less stable than the native state, cannot be formed. The absence of transiently formed folding intermediates in the region of the folding transition has previously been shown for a number of smaller proteins like RNase A (Nall et al, 1978; and RNase T1 .…”

Section: Absence Of Structural Kinetic Intermediatesmentioning

confidence: 58%

“…Folding of the constant fragment of the immunoglobulin light chain was shown to be faster than prolyl isomerization under all conditions with a minimum of about 0.25 s-' for the apparent rate constant of the UF c) N reaction at the midpoint of the folding transition at 25 "C (Goto & Hamaguchi, 1982). In the case of RNase A, folding in the transition region is only slightly faster than prolyl isomerization and thus comparable to aldolase folding (Nall et al, 1978;. Other proteins were shown to fold slowly in the region of the folding transition.…”

Section: Properties Of the Folding Reactionmentioning

confidence: 86%

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Reversible unfolding and refolding behavior of a monomeric aldolase from staphylococcus aureus

1992

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Thermal and GdmC1-induced unfolding transitions of aldolase from Stuphylococcus uureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a AGO value of -9 f 2 kJ/mol at 20 "C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 "C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of a//3 barrel proteins.Keywords: a//3 barrel; folding intermediates; prolyl isomerization; protein folding kinetics; two-state model The mechanism of protein folding has been the subject of intensive studies over the last years. Experimental approaches to the folding problem have mainly focused on the elucidation of folding pathways, the characterization of folding intermediates, and the investigation of the dominant forces in protein stability (for reviews see Jaenicke, 1987;Privalov & Gill, 1988;Kuwajima, 1989;Kim & Baldwin, 1990). It is now widely accepted that protein folding advances through a number of definite intermediate stages (Baldwin, 1990). In the millisecond time region, secondary structural elements and parts of the tertiary contacts are formed (Roder et al., 1988;Udgaonkar & Baldwin, 1988Kuwajima, 1989). Subsequent slow steps are often limited by cis trans isomerization reac- Abbreviations; Aldolase, fructose-l,6-bisphosphate aldolase (EC 4.1.2.13) from Stuphylococcus aureus; GdmC1, guanidinium chloride; N, native state of the protein; U, unfolded protein; UF and Us, fast and slow refolding molecules, respectively; AG, free energy of folding; a, reduced amplitude; X, apparent rate constant; 7, relaxation time.

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Section: Properties Of the Folding Reactionsupporting

confidence: 69%

Section: Absence Of Structural Kinetic Intermediatesmentioning

confidence: 58%

Section: Properties Of the Folding Reactionmentioning

confidence: 86%

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Reversible unfolding and refolding behavior of a monomeric aldolase from staphylococcus aureus

1992

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“…Provided the denaturant concentration is low, the time range of refolding is 10-100 ms over a wide range of pH and temperature (Garel et al, 1976;Hagerman & Baldwin, 1976;Lin & Brandts, 1983). The logarithm of the refolding rate decreases linearly with denaturant concentration: there is roughly a 1,000-fold drop between zero denaturant and the edge of the unfolding transition (Nall et al, 1978;Tsong & Baldwin, 1978). Only the Us species of bovine RNase A refold slowly enough to be measured in manual mixing experiments, which we use here.…”

Section: Refolding Kinetics Of Single Mutants At Pi14 and P93mentioning

confidence: 99%

Cis proline mutants of ribonuclease A. II. Elimination of the slow‐folding forms by mutation

Schultz¹,

Schmid²,

Baldwin³

1992

Protein Science

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Ribonuclease A is known to form an equilibrium mixture of fast-folding (U,) and slow-folding (Us) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, UsII, UsI, and possibly also minor populations of other Us species. The two cis proline residues, P93 and P114, are logical candidates for producing the major Us species after unfolding, by slow cis H trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site-directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of Us species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce Us species; thus, Pro 114 is not required for the formation of at least one of the major Us species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis +trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the Us species of ribonuclease A.The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding. Thus, the unfolding and refolding kinetics of the double mutant indicate that replacement of Pro 93 and Pro 114 has removed the residues responsible for forming the major Us species. This result, taken together with data for the Pro 114 single mutants, indicates that Pro 93 and Pro 114 are collectively responsible for forming the major Us species.

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“…This phase accounted for about 30% of the amplitude of the total fluorescence change and an apparent rate constant greater than the main phase. Double-jump experiments (Brandts et al, 1975;Nall et al, 1978) have often been used under these circumstances t o examine whether two forms of the unfolded species exist in the unfolded state. In the case of RNase T2, however, because the unfolding rate was very slow (complete unfolding requires 2 or 3 min even in 6 M Gdn-HCI), we could not perform the double-jump experiment.…”

Section: Kinetics Of Unfolding and Refoldingmentioning

confidence: 99%

Stability of ribonuclease T2 from Aspergillus oryzae

Kawata

Hamaguchi

1995

Protein Science

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The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mo1-' at 25 "C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the T.,, and the enthalpy change of RNase T2 were found to be 55.3 "C and 119.1 kcal.mol", respectively. From these equilibrium and kinetic studies, it was found that the stability of RNase T2 in the native state is predominantly due to the slow rate of unfolding.Keywords: stability; RNase T2; unfolding and refolding mechanism Ribonuclease (RNase) T2 was first reported by Sat0 and Egami (1957) as a ribonuclease in Taka-diastase produced from a culture extract of Aspergillus oryzae and purified independently by Rushizky and Sober (1963) and Uchida (1966). RNase T2 exerts its catalytic activity on the phosphodiester bonds of RNA with a preference for adenylic acid residues without an absolute base specificity (Rushizky & Sober, 1963). For the reason of this nonspecificity, RNase T2 is often used in molecular genetic studies.RNase T2 is a 29-kDa monomeric glycoprotein composed of 239 amino acid residues and about 8% oligosaccharides and has five disulfide bonds in its tertiary structure (Kawata et al., 1988). Gene cloning of this enzyme was also performed by Ozeki et al. (1991). The catalytic amino acid residues of RNase T2 were found to be His 53 and His 115 by chemical modification and NMR experiments . From the sequence alignment between RNase T2 and other proteins, Kawata et al. (1990) found that high sequence similarities to the regions around the two catalytic histidine residues of RNase T2 could be observed in a 32-kDa S2 stylar glycoprotein (Anderson et al., 1986) from Nicotiana alata, which was expressed in a gametophytic self-incompatible stylar. From this fact, Kawata et al. proposed that the S2 glycoprotein is a kind of RNase. McCIure et al. (1989) subsequently determined that the S2 glycoprotein is an RNase by activity measurements. This enzyme, called S-RNase, was suggested to be very important in the mechanism of gametophytic self-incompatibility in flowering plants (Haring et al., 1990;McClure et al., 1990;Clarke & Newbigin, 1993). Consequently, studies of structural stability and the folding Reprint requests to: Yasushi Kawata, Department of Biotechnology, Faculty of Engineering, Tottori University, Koyama-Minami, Tottori 680, Japan. mechanism of RNase T2 may provide valuable information regarding S-RNase, and ultimately, the mechanism of selfincompatibility. Also, detailed structural characterization of RNase T2 is a prerequisite in studies regarding the catalytic properties of this enzyme.In this paper, we have studied the stability of RNase T2 from A . oryzae against guanidine hydrochlori...

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Test of the extended two-state model for the kinetic intermediates observed in the folding transition of ribonuclease A

Cited by 151 publications

References 19 publications

Reversible unfolding and refolding behavior of a monomeric aldolase from staphylococcus aureus

Reversible unfolding and refolding behavior of a monomeric aldolase from staphylococcus aureus

Cis proline mutants of ribonuclease A. II. Elimination of the slow‐folding forms by mutation

Stability of ribonuclease T2 from Aspergillus oryzae

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