A Native‐Like Intermediate on the Ribonuclease A Folding Pathway

Schmid, Franz X.; Blaschek, Heidi

doi:10.1111/j.1432-1033.1981.tb06180.x

Cited by 117 publications

(8 citation statements)

References 26 publications

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“…Earlier results obtained by spectroscopic measurements indicated that within the first milliseconds of refolding of the slow folding species U S II , an early intermediate (I 1 ) is populated. This intermediate is considered to have a well-defined hydrogen bond network and some of the secondary structural features of the native protein. ,, I 1 then folds to the nativelike intermediate (I N ) which possesses already many properties of the native protein, such as enzymatic activity, binding to the specific inhibitor 2‘-CMP, and a similar tyrosine absorbance. I N is further characterized by at least one non-native peptidyl prolyl bond.…”

Section: Resultsmentioning

confidence: 99%

Isothermal Calorimetry as a Tool To Investigate Slow Conformational Changes in Proteins and Peptides

et al. 2006

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A new calorimetric method has been developed to follow the time course of slow conformational changes during the refolding of denatured proteins. The method is based on the ability of isothermal titration calorimeters (ITC) to detect small amounts of heat continuously over a minute to an hour time range without being disturbed by baseline drift. We benchmarked the method on the basis of the slow kinetic phases resulting from prolyl cis/trans isomerization of oligopeptides. Using this method, the simultaneous investigation of the kinetics and thermodynamics of slow phases in the refolding of GdmCl-denatured RNase A by single jump techniques was performed. Time traces of heat production in the presence of a peptidyl prolyl cis/trans isomerase support the classical model of rate-limiting prolyl trans to cis isomerizations in the folding reactions of RNase A. However, we also observed that, unlike prolyl cis/trans isomerizations in oligopeptides, those found in RNase A refolding are highly exothermic. It appears that coupling between slow prolyl trans to cis isomerization and relocation of remote backbone segments increases the number of contacting sites during formation of the native protein. The results demonstrate that calorimetrically monitored folding kinetics will be of relevance in the detection of otherwise silent folding events.

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Section: Resultsmentioning

confidence: 99%

Isothermal Calorimetry as a Tool To Investigate Slow Conformational Changes in Proteins and Peptides

et al. 2006

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“…The log plot in Fig. 2, representing the slow US --j N reaction, is not strictly linear, suggesting that there may be more than one slow refolding reaction [9,18,19]. A different pattern emerges when refolding of Us is carried out under strongly native conditions, such as pH 6, 0.2 M guanidine .…”

Section: Slow Folding Kinetics Monitored By Diffiwnt Probesmentioning

confidence: 99%

“…There is good evidence that this complexity is due to two slow-folding species U8 and Uk', which exist in a slow equilibrium and give rise to two separate refolding reactions, a faster one, U;' -+ N, with 80 -90 yd relative amplitude and a slower one, Ui -+ N, with 10-20 ; < relative amplitude [9,19]. To correlate folding kinetics measured by different probes it is necessary to confirin that the same folding reactions are used for this reaction) can be ruled out by a direct comparison of both kinetics.…”

Section: Kinetic Hlermediute In the Slow Folding Reaction Of Rnnse Amentioning

confidence: 99%

A Native‐Like Intermediate on the Ribonuclease A Folding Pathway

Schmid

1981

European Journal of Biochemistry

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Unfolded ribonuclease A consists of a mixture of fast-folding (UF) and slow-folding species (US); US folds slowly because it contains non-native proline isomers. The folding kinetics of US have been determined by measuring the changes in tyrosine fluorescence upon refolding. Under conditions where the energy of stabilization of the native protein is small (in marginally native conditions) the fluorescence-detected kinetics of folding coincide with the ones measured by other probes. However, when refolding is carried out under conditions where the native protein is highly stabilized (in strongly native conditions), the fluorescence changes become progressively slower than folding monitored by tyrosine absorbance or by 2'CMP binding. Under these conditions the fluorescence-detected kinetics of folding show properties of proline isomerization reactions. In addition they match proline isomerization measured during refolding of US by a two-step assay for non-native proline isomers.The following conclusions are drawn.(1) There is a kinetic intermediate between US and the native enzyme in the folding pathway of RNase A. This has been shown by the divergent progress curves monitored by different probes. Although the intermediate contains non-native proline isomers, it is folded and hence sensitive to agents that affect the stability of native proteins. (2) Fluorescence-detected folding closely follows proline isomerization during folding. Refolding of US is not blocked by incorrect proline isomers, but US is able to fold to a structural intermediate before proline isomerization occurs. (3) The quantum yield of the fluorescence of native RNase A depends on the precise spatial location of the tyrosine side chains with respect to their hydrogen bond acceptors and to the disulfide bonds. Therefore the native fluorescence is not regained until all essential proline peptide bonds are in the right configuration.There is now increasing evidence that folding of small proteins proceeds on an ordered pathway, the rate of folding being controlled by detectable intermediates [l]. In order to elucidate the folding mechanism of a particular protein it is necessary to characterize these intermediates and place them in the right order on the folding pathway.

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“…However, in a few cases folding through stable intermediates is debated and questioned [11,12]. Intermediates have been reported to arise as a consequences of energy barrier [13], incorrect proline-isomerization [14,15], non native disulfide bond formation [16], interaction with cofactors such as hemegroup [17] or non-native contact that need to be broken before folding proceeds [18]. An understanding of the structural and thermodynamic properties of such intermediate states may give an insight into these various factors involved in guiding the pathway for folding of the protein.…”

Section: Folding Intermediatementioning

confidence: 99%

Snapshots of Protein Folding Problem: Implications of Folding and Misfolding Studies

Dubey¹,

Pande²,

Jagannadham

2006

PPL

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Deciphering the code that determines the three-dimensional structure of proteins and the ability to predict the final folded form of a protein is still elusive to molecular biophysists. In the case of several proteins a similar tertiary structure is not accompanied by any significant sequence similarity. The question now remains whether a code beyond the genetic code that describes the arrangement of the amino acid within a three dimensional protein structure. The available data undoubtedly demonstrates that the redundancy of this code must be tremendous. Several techniques such as nuclear magnetic resonance spectroscopy and laser detection techniques, coupled with fast initiation of the folding reaction, can now probe the folding events in milliseconds or even faster and provide highly relevant information. The thermodynamic analysis of the folding process and of kinetic intermediates opens whole new avenue of understanding. Breaking the protein folding code would enable scientists to look at a gene whose function is unknown and predict the three-dimensional structure of the protein it encodes. This would give them a very good idea of what the gene does. In this review we hope to bring together the information available about protein folding with particular emphasis on folding intermediate(s). Additionally, the practical consequences of the solution of the protein folding problem in medicine and biotechnology are also discussed.

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A Native‐Like Intermediate on the Ribonuclease A Folding Pathway

Cited by 117 publications

References 26 publications

Isothermal Calorimetry as a Tool To Investigate Slow Conformational Changes in Proteins and Peptides

Isothermal Calorimetry as a Tool To Investigate Slow Conformational Changes in Proteins and Peptides

A Native‐Like Intermediate on the Ribonuclease A Folding Pathway

Snapshots of Protein Folding Problem: Implications of Folding and Misfolding Studies

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