Folding of ribonuclease T1. 1. Existence of multiple unfolded states created by proline isomerization.
It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.
Kiefhaber
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Laboratorium für Biochemie, Universität Bayreuth, West Germany.
Quaas
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Hahn
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Schmid
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D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007536:Isomerism; D007700:Kinetics; D011392:Proline; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification; D006163:Ribonuclease T1 / Q000235:genetics / Q000302:isolation & purification; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D011994:Recombinant Proteins; D014508:Urea; D011392:Proline; D004722:Endoribonucleases; D006163:Ribonuclease T1; D019791:Guanidine