Stopped flow dynamic light scattering as a method to monitor compaction during protein folding

被引:34
作者
Gast, K [1 ]
Noppert, A [1 ]
MullerFrohne, M [1 ]
Zirwer, D [1 ]
Damaschun, G [1 ]
机构
[1] HUMBOLDT UNIV BERLIN,INST BIOL,MAX DELBRUCK CTR MOL MED,D-13122 BERLIN,GERMANY
来源
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS | 1997年 / 25卷 / 03期
关键词
protein folding; dynamic light scattering; stopped-flow kinetics; ribonuclease A; phosphoglycerate kinase;
D O I
10.1007/s002490050033
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
Kinetic dynamic light scattering is a useful tool to follow compaction during protein folding. In contrast to measurements of the formation of secondary structure and side chain ordering, kinetic measurements of compactness are not well established up to now. This work describes the adaptation of a stopped-flow system (SFM-3) to a dynamic light scattering apparatus, which allows one to monitor the compaction of protein molecules by measuring the hydrodynamic Stokes radius R. The feasibility of such investigations was demonstrated by measuring R and the integrated scattered intensity I during refolding of ribonuclease A and phosphoglycerate kinase from yeast. Refolding was initiated by rapid mixing of protein solutions containing high concentrations of guanidine hydrochloride with buffer. Between 20 and 50 mixing events were performed in these experiments. Measuring both R and I in one and the same experiment is important to distinguish between true folding of individual molecules and cases where folding is accompanied by the appearance of transient oligomers or associated misfolded structures. On refolding of ribonuclease A (0.6 M GuHCl, 25 degrees C), after a fast phase the Stokes radius decreased from 2.26 nm to 1.95 nm with a time constant of 27 s without detectable aggregates. By contrast, transient and stable oligomers have been observed during the more complex folding of phosphoglycerate kinase. In general, the time-resolution of the method is of the order of 1 s. It can be extended to the subsecond time-range if the number of shots is not limited by the amount of protein available.
引用
收藏
页码:211 / 219
页数:9
相关论文
共 49 条
[11]   Folding and unfolding kinetics of the proline-to-alanine mutants of bovine pancreatic ribonuclease A [J].
Dodge, RW ;
Scheraga, HA .
BIOCHEMISTRY, 1996, 35 (05) :1548-1559
[12]   EVIDENCE OF AN ASSOCIATIVE INTERMEDIATE ON THE MYOGLOBIN REFOLDING PATHWAY [J].
ELIEZER, D ;
CHIBA, K ;
TSURUTA, H ;
DONIACH, S ;
HODGSON, KO ;
KIHARA, H .
BIOPHYSICAL JOURNAL, 1993, 65 (02) :912-917
[13]   THE RADIUS OF GYRATION OF AN APOMYOGLOBIN FOLDING INTERMEDIATE [J].
ELIEZER, D ;
JENNINGS, PA ;
WRIGHT, PE ;
DONIACH, S ;
HODGSON, KO ;
TSURUTA, H .
SCIENCE, 1995, 270 (5235) :487-488
[14]   EARLY STEPS IN CYTOCHROME-C FOLDING PROBED BY TIME-RESOLVED CIRCULAR-DICHROISM AND FLUORESCENCE SPECTROSCOPY [J].
ELOVE, GA ;
CHAFFOTTE, AF ;
RODER, H ;
GOLDBERG, ME .
BIOCHEMISTRY, 1992, 31 (30) :6876-6883
[15]   LIFETIME OF THE HISTONE OCTAMER STUDIED BY CONTINUOUS-FLOW QUASI-ELASTIC LIGHT-SCATTERING - TEST OF A MODEL FOR NUCLEOSOME TRANSCRIPTION [J].
FENG, HP ;
SCHERL, DS ;
WIDOM, J .
BIOCHEMISTRY, 1993, 32 (30) :7824-7831
[16]   KINETICS OF COMPACTION DURING LYSOZYME REFOLDING STUDIED BY CONTINUOUS-FLOW QUASI-ELASTIC LIGHT-SCATTERING [J].
FENG, HP ;
WIDOM, J .
BIOCHEMISTRY, 1994, 33 (45) :13382-13390
[17]   TRUNCATED STAPHYLOCOCCAL NUCLEASE IS COMPACT BUT DISORDERED [J].
FLANAGAN, JM ;
KATAOKA, M ;
SHORTLE, D ;
ENGELMAN, DM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (02) :748-752
[18]   BOTH FAST AND SLOW REFOLDING REACTIONS OF RIBONUCLEASE-A YIELD NATIVE ENZYME [J].
GAREL, JR ;
BALDWIN, RL .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1973, 70 (12) :3347-3351
[19]  
GAST K, 1992, EUR BIOPHYS J BIOPHY, V21, P357, DOI 10.1007/BF00188349
[20]   COMPACTNESS OF PROTEIN MOLTEN GLOBULES - TEMPERATURE-INDUCED STRUCTURAL-CHANGES OF THE APOMYOGLOBIN FOLDING INTERMEDIATE [J].
GAST, K ;
DAMASCHUN, H ;
MISSELWITZ, R ;
MULLERFROHNE, M ;
ZIRWER, D ;
DAMASCHUN, G .
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, 1994, 23 (04) :297-305