Variable velocity liquid flow EPR applied to submillisecond protein folding

Biophys J. 2000 May;78(5):2702-8. doi: 10.1016/S0006-3495(00)76814-7.

Abstract

We have developed a variable velocity, rapid-mix, continuous-flow method for observing and delineating kinetics by dielectric resonator-based electron paramagnetic resonance (EPR). The technology opens a new facet for kinetic study of radicals in liquid at submillisecond time resolution. The EPR system (after Sienkiewicz, A., K. Qu, and C. P. Scholes. 1994. Rev. Sci. Instrum. 65:68-74) accommodated a miniature quartz capillary mixer with an approximately 0.5 microliter delivery volume to the midpoint of the EPR-active zone. The flow velocity was varied in a preprogrammed manner, giving a minimum delivery time of approximately 150 microseconds. The mixing was efficient, and we constructed kinetics in the 0.15-2. 1-ms time range by plotting the continuous wave EPR signal taken during flow versus the reciprocal of flow velocity. We followed the refolding kinetics of iso-1-cytochrome c spin-labeled at Cysteine 102. At 20 degrees C, upon dilution of guanidinium hydrochloride denaturant, a fast phase of refolding was resolved with an exponential time constant of 0.12 ms, which was consistent with the "burst" phase observed by optically detected flow techniques. At 7 degrees C the kinetic refolding time of this phase increased to 0.5 ms.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biophysical Phenomena
  • Biophysics
  • Cyclic N-Oxides
  • Cysteine / chemistry
  • Cytochrome c Group / chemistry
  • Cytochromes c*
  • Electron Spin Resonance Spectroscopy / instrumentation
  • Electron Spin Resonance Spectroscopy / methods*
  • Kinetics
  • Mesylates
  • Protein Folding*
  • Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins*
  • Spin Labels

Substances

  • CYC1 protein, S cerevisiae
  • Cyclic N-Oxides
  • Cytochrome c Group
  • Mesylates
  • Proteins
  • Saccharomyces cerevisiae Proteins
  • Spin Labels
  • (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate
  • Cytochromes c
  • Cysteine