In erythrocytes, 4.1R80 (80 kDa isoform of protein 4.1R) binds to the cytoplasmic tail of the transmembrane proteins band 3 and GPC (glycophorin C), and to the membrane-associated protein p55 through the N- (N-terminal), α- (α-helix-rich) and C- (C-terminal) lobes of R30 [N-terminal 30 kDa FERM (4.1/ezrin/radixin/moesin) domain of protein 4.1R] respectively. We have shown previously that R30 binds to CaM (calmodulin) in a Ca2+-independent manner, the equilibrium dissociation constant (Kd) for R30-CaM binding being very similar (in the submicromolar range) in the presence or absence of Ca2+. In the present study, we investigated the consequences of CaM binding on R30's structural stability using resonant mirror detection and FTIR (Fourier-transform IR) spectroscopy. After a 30 min incubation above 40° C, R30 could no longer bind to band 3 or to GPC. In contrast, R30 binding to p55, which could be detected at a temperature as low as 34° C, was maintained up to 44° C in the presence of apo-CaM. Dynamic light scattering measurements indicated that R30, either alone or complexed with apo-CaM, did not aggregate up to 40° C. FTIR spectroscopy revealed that the dramatic variations in the structure of the β-sheet structure of R30 observed at various temperatures were minimized in the presence of apo-CaM. On the basis of Kd values calculated at various temperatures, ΔCp and ΔG° for R30 binding to apo-CaM were determined as -10 kJ · K(-1) · mol-1 and ~ -38 kJ · mol(-1) at 37° C (310.15 K) respectively. These data support the notion that apo-CaM stabilizes R30 through interaction with its β-strand-rich C-lobe and provide a novel function for CaM, i.e. structural stabilization of 4.1R80.

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detergents induce alpha-helix formation irrespective of the intrinsic native secondary structure, a process known as "reconstructive denaturation." Although this latter phenomenon underpins the ubiquitous technique SDS-PAGE, the mechanism of SDS denaturation and the molecular nature of the SDS denatured state are not known. We use a combined biophysical and computational approach to elucidate the molecular basis of protein denaturation by ionic detergents, with a special focus on the mechanism of reconstructive denaturation by SDS. Specifically, biophysical techniques, including CD and ITC, are used to study the interaction of a set of detergents with model peptides in parallel with molecular dynamics simulations of the same systems. Our results show that SDS and LTAC induce increased alpha-helix content in cationic and anionic peptides respectively, but not vice versa. The zwitterionic detergent lauryl-dimethylamine oxide (LDAO) has no effect on either peptide. Our MD simulations provide atomic resolution detail of the results from the biophysical experiments, and show different modes of micellar binding that correlate with the observed detergent/peptide data. These results suggest a mechanism for the reconstructive denaturation phenomenon and for SDS's universal protein denaturing action.