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

Marco De Spirito, Francesco Ferri, F. Ferri

Risultato della ricerca: Contributo in rivistaArticolo in rivista

1 Citazioni (Scopus)

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 Rs. 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.
Lingua originaleEnglish
pagine (da-a)205-220
Numero di pagine16
RivistaMacromolecular Symposia
Volume162
DOI
Stato di pubblicazionePubblicato - 2000

Keywords

  • EMTREE drug terms: alpha lactalbumin
  • phosphoglycerate kinase
  • ribonuclease A

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