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Cooperative -helix unfolding in a protein–DNA complex from hydrogen–deuterium exchange

Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands

(RECEIVED November 1, 2005; FINAL REVISION February 16, 2006; ACCEPTED March 7, 2006)

We present experimental evidence for a cooperative unfolding transition of an -helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen–deuterium (H–D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton H^N to exchange. Close to this regime, we measured decay rates of individual backbone H^N signals in D₂O, and of their mutual H^N–H^N NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20–Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that H^N exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the H^N exchange in helix 3 is dominated by random local structure fluctuations.

Keywords: hydrogen–deuterium exchange; folding; cooperativity; protein–DNA complex; Lac repressor

Supplemental material: see www.proteinscience.org

Reprint requests to: Rolf Boelens, Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; e-mail: r.boelens{at}chem.uu.nl; fax: +31-30-253-7623.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051938006.

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