首页 | 本学科首页   官方微博 | 高级检索  
     


Test of the extended two-state model for the kinetic intermediates observed in the folding transition of ribonuclease A
Authors:Barry T. Nall  Jean-Renaud Garel  Robert L. Baldwin
Affiliation:1. Department of Biochemistry Stanford University Medical School Stanford, Calif. 94305, U.S.A.;2. Service de Biochimie Cellulaire Institut Pasteur Paris 75724, France
Abstract:
A test has been made of the proposal that: (a) the extended two-state model describes the kinetic intermediates seen in the folding transition of RNAase A, i.e. that the only species present in folding experiments are the native protein and multiple forms of the completely unfolded protein; and (b) that the interconversion between the two known unfolded forms of RNAase A (the U1
/></figure>U<sub>2</sub> reaction) is described solely by the <em>cis-trans</em> isomerization of the proline residues. The test is to measure the rate of the U<sub>1</sub><figure class=/></figure>U<sub>2</sub> reaction in a wide range of refolding conditions and to compare these data with the kinetic properties of proline isomerization.The main results are as follows. (1) The activation enthalpy of the U<sub>1</sub><figure class=/></figure>U<sub>2</sub> reaction in refolding conditions (pH 6, 20 ° to 40 °C) is less than 5 kcal/mol. This is much too small to be explained as proline isomerization. (2) Both the rate and the activation enthalpy change sharply at guanidine hydrochloride concentrations below 2 m. There appear to be two pathways for the U<sub>1</sub><figure class=/></figure>U<sub>2</sub> reaction in refolding conditions, and the slower pathway is favored by adding guanidine hydrochloride. (3) The rate and activation enthalpy for proline isomerization in l-alanyl-l-proline are unaffected by 2 m-guanidine hydrochloride.The results show that the proline isomerization hypothesis and the extended two-state model cannot both be correct for RNAase A. They suggest that partial folding occurs rapidly in refolding conditions and that the extended two-state model is invalid. They leave open the question of whether or not proline isomerization is the rate-limiting step in the U<sub>1</sub><figure class=/></figure>U<sub>2</sub> reaction.Another possible source of slow configurational reactions in the unfolded state is mentioned. The three major, overlapping, disulfide-bonded loops of RNAase A can exist in two isomeric configurations. Interconversion of these isomers requires pulling one loop, or one end of the polypeptide chain, through a second loop and this is likely to be a slow process.In some conditions, heat-unfolded but not guanidine-unfolded RNAase A shows a second slow-refolding process. It may result from aggregates of the heatunfolded protein which are formed and broken up slowly. Conditions are given for eliminating this reaction.</td> </tr> <tr> <td align=
Keywords:To whom correspondence should be adressed
本文献已被 ScienceDirect 等数据库收录！
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号