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Biophysical Journal 84:533-544 (2003)
© 2003 The Biophysical Society

Cooperativity in Forced Unfolding of Tandem Spectrin Repeats

Richard Law*, Philippe Carl*, Sandy Harper{dagger}, Paul Dalhaimer*, David W. Speicher*,{dagger} and Dennis E. Discher*,{dagger}

* Biophysical Engineering Lab, Institute for Medicine and Engineering; and School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6315, and {dagger}Structural Biology Program, The Wistar Institute, Philadelphia, Pennsylvania 19104

Correspondence: Address reprint requests to Dennis E. Discher, Biophysical Engineering Lab, 112 Towne Building, University of Pennsylvania, Philadelphia, PA 19104-6315. Tel.: 215-898-4809; Fax: 215-573-6334; E-mail: discher{at}seas.upenn.edu.

Force-driven conformational changes provide a broad basis for protein extensibility, and multidomain proteins broaden the possibilities further by allowing for a multiplicity of forcibly extended states. Red cell spectrin is prototypical in being an extensible, multidomain protein widely recognized for its contribution to erythrocyte flexibility. Atomic force microscopy has already shown that single repeats of various spectrin family proteins can be forced to unfold reversibly under extension. Recent structural data indicates, however, that the linker between triple-helical spectrin repeats is often a contiguous helix, thus raising questions as to what the linker contributes and what defines a domain mechanically. We have examined the extensible unfolding of red cell spectrins as monomeric constructs of just two, three, or four repeats from the actin-binding ends of both {alpha}- and ß-chains, i.e., {alpha}18–21 and ß1–4 or their subfragments. In addition to single repeat unfolding evident in sawtooth patterns peaked at relatively low forces (<50 pN at 1 nm/ms extension rates), tandem repeat unfolding is also demonstrated in ensemble-scale analyses of thousands of atomic force microscopy contacts. Evidence for extending two chains and loops is provided by force versus length scatterplots which also indicate that tandem repeat unfolding occurs at a significant frequency relative to single repeat unfolding. Cooperativity in forced unfolding of spectrin is also clearly demonstrated by a common force scale for the unfolding of both single and tandem repeats.




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