Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Tsuyoshi Konuma, Tetsunari Kimura, Shuzo Matsumoto, Yuji Goto, Tetsuro Fujisawa, Alan R. Fersht, Satoshi Takahashi

Research output: Contribution to journalArticlepeer-review

36 Citations (Scopus)

Abstract

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, Dphys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (Rg) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the Rg value extrapolated from kinetic Rg data to time zero, Rg,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in Rg2 was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated Rg of the minor component of Dphys containing the non-native cis proline isomer (Dphys,cis) to be 25.7 ± 0.6 Å. Moreover, Rg of the major component of D phys containing the native proline isomer (Dphys,tra) was estimated as 23.9 ± 0.2 Å based on Rg,0. Consequently, both components of the burst-phase intermediate of barnase (Dphys,tra and Dphys,cis) are still largely expanded. It was inferred that Dphys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.

Original languageEnglish
Pages (from-to)1284-1294
Number of pages11
JournalJournal of Molecular Biology
Volume405
Issue number5
DOIs
Publication statusPublished - 2011 Feb 4

Keywords

  • barnase
  • protein folding
  • small-angle X-ray scattering

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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