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Published online before print June 2, 2006, 10.1110/ps.051938006
Protein Science (2006), 15:1752-1759. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Cooperative {alpha}-helix unfolding in a protein–DNA complex from hydrogen–deuterium exchange

Roberto K. Salinas1, Tammo Diercks, Robert Kaptein and Rolf Boelens

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 {alpha}-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 HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN–HN 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 HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.

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


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