To understand and tackle amyloid-related diseases, it is crucial to investigate the factors that modulate amyloid formation of proteins. Our previous studies proved that the N47A mutant of the α-spectrin SH3 (Spc-SH3) domain forms amyloid fibrils quickly under mildly acidic conditions. Here, we analyze how experimental conditions influence the kinetics of assembly and the final morphology of the fibrils. Early formation of curly fibrils occurs after a considerable conformational change of the protein and the concomitant formation of small oligomers. These processes are strongly accelerated by an increase in salt concentration and temperature, and to a lesser extent by a reduction in pH. The rate-limiting step in these events has a high activation enthalpy, which is significantly reduced by an increase in NaCl concentration. At low-to-moderate NaCl concentrations, the curly fibrils convert to straight and twisted amyloid fibrils after long incubation times, but only in the presence of soluble species in the mixture, which suggests that the curly fibrils and the twisted amyloid fibrils are diverging assembly pathways. The results suggest that the influence of environmental variables on protein solvation is crucial in determining the nucleation kinetics, the pathway of assembly, and the final fibril morphology.

The activation domain of human procarboxypeptidase A2 (ADA2h), a globular open-sandwich alpha + beta domain with 80 residues and no disulfide bridges, has been studied by thermodynamic and kinetic analysis. Equilibrium denaturation by urea or temperature is fully reversible at pH 7.0 and fits to a two-state transition. The Gibbs energy of unfolding extrapolated to null concentration of chemical denaturant, delta GH2O, at pH 7.0 and 298 K, is calculated to be 17.0 +/- 1 kJ mol-1, which is within experimental error of the value determined by differential scanning calorimetry, 15.1 +/- 2 kJ mol-1. Kinetics of unfolding and refolding followed by fluorescence do not show the presence of any kinetic intermediate accumulating in the folding reaction. A value for delta GH2O of 17.9 +/- 0.7 kJ mol-1 can be extrapolated from the kinetic data. All these data indicate that the folding pathway of this domain is consistent with a two-state model (with the exception of the cis-Pro intermediates). More importantly, the analysis of this and several other small domains or proteins supports the hypothesis that stable kinetic folding intermediates are not necessary for a protein to fold. There seems to be a relationship between the size of a protein and the presence of stable kinetic intermediates. Globular proteins with less than 80 residues and no disulfide bonds follow a two-state transition, while proteins larger than 100 residues present stable kinetic folding intermediates.

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The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 μM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd = 132 ± 13 μM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes.