Time-resolved dynamic light scattering as a method to monitor compaction during protein folding

The mechanisms and the dynamics of protein folding are subject of a still i ncreasing number of theoretical and experimental studies. While spectroscop ic methods are already used for many years to measure the folding rates and to monitor the formation of secondary and tertiary structure, kinetic meas urements of the compactness are only beginning to emerge. Time-resolved dyn amic light scattering (DLS) is a useful tool to follow the compaction durin g protein folding by measuring the hydrodynamic Stokes radius R-S. Addition ally changes in the state of association can be detected by simultaneous me asurements of the scattering intensity. The usefulness of different techniq ues for time-resolved DLS measurements and the general limits for kinetic D LS experiments are discussed first. Then we describe the adaptation of a st opped-flow system (SFM-3) to a DLS apparatus, the particular data acquisiti on schemes, and the experimentally attainable limits. The feasibility of st opped-flow DLS is demonstrated by the results of folding investigations wit h ribonuclease A, phosphoglycerate kinase, and bovine alpha -lactalbumin. R efolding was initiated by denaturant dilution jumps, which were repeated up to 100 times in order to obtain a reasonable signal-to-noise ratio. Kineti c DLS experiments can be performed fairly with a time resolution of one sec ond. The time resolution of 100ms is probably the attainable limit. The cap abilities of time-resolved DLS and time-resolved small-angle X-ray scatteri ng are compared.