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The expected contour length increase upon unfolding of a protein domain consisting of N amino acid residues can be calculated as ΔL, 0.365N, dfolded(I, dfolded(II, where dfolded(I) and dfolded(II) are the end-to-end distances of the folded structure before and after the unfolding event, respectively SOM text, Due to the a-helical linker between DomN and DomC, the domain boundaries of CaM are not precisely defined, which may account for the small deviations from the expected values
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folded(II) are the end-to-end distances of the folded structure before and after the unfolding event, respectively (SOM text). Due to the a-helical linker between DomN and DomC, the domain boundaries of CaM are not precisely defined, which may account for the small deviations from the expected values.
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A simple thermodynamic argument can readily explain why a 1:1 stoichiometry should lead to concentration independent forces and transition rates (dashed lines in Fig. 3B). In our assay, the fully folded CaM is transformed to CaM with unfolded DomN in the first unfolding peak. Because at all Mas concentrations used (>0.3 mM), both intact CaM and DomC alone are fully saturated with Mas, changes in concentration cannot affect the energetics of this transition any further (SOM text).
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We thank P. M. Bayley for inspiring discussions and A. Bausch, H. Gaub, M. Bertz, and M. Schlierf for helpful comments on the manuscript. This work was supported by Deutsche Forschungsgemeinschaft grant RI 990/3-1. J.P.J. was supported by the International Graduate School Materials Science of Complex Interfaces.
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We thank P. M. Bayley for inspiring discussions and A. Bausch, H. Gaub, M. Bertz, and M. Schlierf for helpful comments on the manuscript. This work was supported by Deutsche Forschungsgemeinschaft grant RI 990/3-1. J.P.J. was supported by the International Graduate School "Materials Science of Complex Interfaces."
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