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Analysis of subsecond protein dynamics by amide hydrogen exchange and mass spectrometry using a quenched-flow setup

¹ Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, 69120 Heidelberg, Germany
² Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark

Amide hydrogen exchange (HX) in combination with mass spectrometry (MS) is a powerful tool to analyze the folding and dynamics of proteins. In the traditional methodology the exchange time is controlled by manual pipetting, thereby limiting the time resolution to several seconds. Some conformational changes in proteins, however, occur in the subsecond time scale, making it desirable to perform HX at shorter time intervals down to the limit set by the intrinsic chemical exchange rate. We now report the development of the first completely on-line quenched-flow setup that allows the performance of HX experiments in the 100-sec to 30-sec time scale, on-line proteolytic digestion using immobilized proteases, rapid desalting, and MS analysis. We show that conformational fluctuations in the range of seconds can be detected and protection factors as small as 10 reproducibly determined. Using this setup we investigated the conformational properties of Escherichia coli heat-shock transcription factor ³² free in solution. Our results indicate that the C-terminal ₄ domain of ³², which is responsible for the recognition of the –35 region of heat shock promoters, contains more extensive secondary structure than expected when compared with the structure of the homologous -factor ^A in complex with the RNA-polymerase. This setup should be very useful for a more accurate analysis of structural motions in proteins in the subsecond to second time scale relevant to allostery and enzyme function.

Keywords: quenched flow; amide hydrogen exchange; protein conformation; protein folding; mass spectrometry

Abbreviations: ESI, electrospray ionization • HPLC, high-pressure liquid chromatography • HX, amide hydrogen-(¹H/²H)-exchange • k_ch, intrinsic chemical exchange rate • k_obs, observed exchange rate • MS, mass spectrometry

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041098305.

Reprint requests to: Matthias P. Mayer, Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany; e-mail: M.Mayer{at}zmbh.uni-heidelberg.de; fax: 49 6221 54 5894.

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