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BIOLOGICAL SCIENCES / BIOPHYSICS
Predicting coupling limits from an experimentally determined energy landscape

Timothy O. Street, Christina M. Bradley, and Doug Barrick

T. C. Jenkins Department of Biophysics, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218

Edited by S. Walter Englander, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved January 11, 2007 (received for review October 5, 2006)

Repeat proteins are composed of tandem structural modules in which close contacts do not extend beyond adjacent repeats. Despite the local nature of these close contacts, repeat proteins often unfold as a single, highly coupled unit. Previous studies on the Notch ankyrin domain suggest that this lack of equilibrium unfolding intermediates results both from stabilizing interfaces between each repeat and from a roughly uniform distribution of stability across the folding energy landscape. To investigate this idea, we have generated 15 variants of the Notch ankyrin domain with single and multiple destabilizing substitutions that make the energy landscape uneven. By applying a free energy additivity analysis to these variants, we quantified the destabilization threshold over which repeats 6 and 7 decouple from repeats 1–5. The free energy coupling limit suggested by this additivity analysis (4 kcal/mol) is also reflected in m-value analysis and in differences among equilibrium unfolding transitions as monitored by CD versus fluorescence for all 15 variants. All of these observations are quantitatively predicted by analyzing the response of the experimentally determined energy landscape to increasing unevenness. These results highlight the importance of a uniform distribution of local stability in achieving cooperative unfolding.

ankyrin repeat | free-energy additivity | multistate unfolding | protein folding

Present address: U.S. Patent and Trademark Office, 600 Dulaney Street, Alexandria, VA 22314.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Here we define local stability to include contributions from both intrinsic and nearest-neighbor interfacial interactions.

To whom correspondence should be addressed. E-mail: barrick{at}jhu.edu

© 2007 by The National Academy of Sciences of the USA

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