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Time‐resolved dynamic light scattering as a method to monitor compaction during protein folding
Author(s) -
Gast Klaus,
Zirwer Dietrich,
Damaschun Gregor
Publication year - 2000
Publication title -
macromolecular symposia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.257
H-Index - 76
eISSN - 1521-3900
pISSN - 1022-1360
DOI - 10.1002/1521-3900(200012)162:1<205::aid-masy205>3.0.co;2-d
Subject(s) - dynamic light scattering , folding (dsp implementation) , scattering , light scattering , chemistry , kinetic energy , hydrodynamic radius , materials science , optics , physics , nanotechnology , nanoparticle , classical mechanics , electrical engineering , engineering
Abstract The mechanisms and the dynamics of protein folding are subject of a still increasing number of theoretical and experimental studies. While spectroscopic methods are already used for many years to measure the folding rates and to monitor the formation of secondary and tertiary structure, kinetic measurements of the compactness are only beginning to emerge. Time‐resolved dynamic light scattering (DLS) is a useful tool to follow the compaction during protein folding by measuring the hydrodynamic Stokes radius R S . Additionally, changes in the state of association can be detected by simultaneous measurements of the scattering intensity. The usefulness of different techniques for time‐resolved DLS measurements and the general limits for kinetic DLS experiments are discussed first. Then we describe the adaptation of a stopped‐flow system (SFM‐3) to a DLS apparatus, the particular data acquisition schemes, and the experimentally attainable limits. The feasibility of stopped‐flow DLS is demonstrated by the results of folding investigations with ribonuclease A, phosphoglycerate kinase, and bovine α‐lactalbumin. Refolding was initiated by denaturant dilution jumps, which were repeated up to 100 times in order to obtain a reasonable signal‐to‐noise ratio. Kinetic DLS experiments can be performed fairly with a time resolution of one second. The time resolution of 100ms is probably the attainable limit. The capabilities of time‐resolved DLS and time‐resolved small‐angle X‐ray scattering are compared.