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A study was done on the downfield-shifted amide protons in recombinant cardiac troponin C (TnC). The hydrogen bond was located by isotope labeling and multidimensional NMR between the amide of Gly70 and the carboxylate side chain of Asp65 even without the bound Ca2+ in the N-terminal of cTnC3. The Ca2+-binding loop is tightened by the binding of Ca2+ at site II which reorients the N-terminal domain beta-sheet. These conformational changes were identified to be responsible for the relative movement of the helices within the troponin complex.

Research was conducted to determine the solution structure of cTnC(81-161) both free and bound to cTnI(33-80). Comparison of structures suggests that the free and bound structures are qualitatively similar but demonstrate some particular differences, which are mainly an opening of the structure and movement of the loop region between helices F and G in the bound form. The residues were shown to be highly conserved which indicates a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.

A docking-and-alignment protocol was devised in order to build amyloid protofilaments of Transthyretin (TTR), starting from partially disrupted TTR monomeric subunits and based on experimentally available information. The docking approach is driven by a combination of shape complementarity and energetic criteria, and uses constraints derived from experimental data obtained for the fibrillar state. The dimeric structures obtained were then subjected to an alignment scheme followed by clustering analysis, producing a collection of protofilaments with distinct geometric properties. The selected protofilament model presented here does agree with known experimental data and general amyloid properties; it is formed by two extended continuous β-sheets with the β-strands perpendicular to the main axis of the protofilament and a helical twist with a period of ∼48 β-strands. This TTR proto-filament model may be an important step in the understanding of the molecular mechanisms of TTR aggregation, as well as, a valuable instrument in drug design strategies against amyloid diseases. [ABSTRACT FROM AUTHOR]

The role of disulfide bridges in the structure, stability, and folding pathways of proteins has been the subject of wide interest in the fields of protein design and engineering. However, the relative importance of entropic and enthalpic contributions for the stabilization of proteins provided by disulfides is not always clear. Here, we perform a detailed analysis of the role of disulfides in the conformational stability of human Interleukin-4 (IL4), a four-helix bundle protein. In order to evaluate the contribution of two out of the three disulfides to the structure and stability of IL4, two IL4 mutants, C3T-IL4 and C24T-IL4, were used. NMR and ANS binding experiments were compatible with altered dynamics and an increase of the nonpolar solvent-accessible surface area of the folded state of the mutant proteins. Chemical and thermal unfolding experiments followed by fluorescence and circular dichroism revealed that both mutant proteins have lower conformational stability than the wild-type protein. Transition temperatures of unfolding decreased 14°C for C3T-IL4 and 10°C for C24T-IL4, when compared to WT-IL4, and the conformational stability, at 25°C, decreased 4.9 kcal/mol for C3T-IL4 and 3.2 kcal/mol for C24T-IL4. Interestingly, both the enthalpy and the entropy of unfolding, at the transition temperature, decreased in the mutant proteins. Moreover, a smaller change in heat capacity of unfolding was also observed for the mutants. Thus, disulfide bridges in IL4 play a critical role in maintaining the thermodynamic stability and core packing of the helix bundle. [ABSTRACT FROM AUTHOR]