A protocol to measure slow protein dynamics of the cholera toxin B pentamers using broadband dielectric spectroscopy.

The present protocol describes how to measure experimentally the slow protein dynamics that take place upon the thermal unfolding of the B subunit cholera toxin pentamers using broadband dielectric spectroscopy (BDS) in weakly hydrated and nanoconfined conditions. Transient unfolding intermediates, rarely identified otherwise, are revealed thanks to the B subunit's remarkable heat resistance up to 180°C and distinct molecular dynamics. The frequencies detected experimentally are consistent with the spatiotemporal scales of motions of molecular dynamics simulation. For complete details on the use and execution of this protocol, please refer to Bourgeat et al. (2021, 2019).

Experimental diagnostic of sequence-variant dynamic perturbations revealed by broadband dielectric spectroscopy.

Genetic diversity leads to protein robustness, adaptability, and failure. Some sequence variants are structurally robust but functionally disturbed because mutations bring the protein onto unfolding/refolding routes resulting in misfolding diseases (e.g., Parkinson). We assume dynamic perturbations introduced by mutations foster the alternative unfolding routes and test this possibility by comparing the unfolding dynamics of the heat-labile enterotoxin B pentamers and the cholera toxin B pentamers, two pentamers structurally and functionally related and robust to 17 sequence variations. The B-subunit thermal unfolding dynamics are monitored by broadband dielectric spectroscopy in nanoconfined and weakly hydrated conditions. Distinct dielectric signals reveal the different B-subunits unfolding dynamics. Combined with network analyses, the experiments pinpoint the role of three mutations A1T, E7D, and E102A, in diverting LTB5 to alternative unfolding routes that protect LTB5 from dissociation. Altogether, the methodology diagnoses dynamics faults that may underlie functional disorder, drug resistance, or higher virulence of sequence variants.

Experimental Protein Molecular Dynamics: Broadband Dielectric Spectroscopy coupled with nanoconfinement.

Protein dynamics covers multiple spatiotemporal scale processes, among which slow motions, not much understood even though they are underlying protein folding and protein functions. Protein slow motions are associated with structural heterogeneity, short-lived and poorly populated conformations, hard to detect individually. In addition, they involve collective motions of many atoms, not easily tracked by simulation and experimental devices. Here we propose a biophysical approach, coupling geometrical nanoconfinement and broadband dielectric spectroscopy (BDS), which distinguishes protein conformations by their respective molecular dynamics. In particular, protein-unfolding intermediates, usually poorly populated in macroscopic solutions are detected. The protein dynamics is observed under unusual conditions (sample nanoconfinement and dehydration) highlighting the robustness of protein structure and protein dynamics to a variety of conditions consistent with protein sustainability. The protein dielectric signals evolve with the temperature of thermal treatments indicating sensitivity to atomic and molecular interaction changes triggered by the protein thermal unfolding. As dipole fluctuations depend on both collective large-scale motions and local motions, the approach offers a prospect to track in-depth unfolding events.

In proteins, the structural responses of a position to mutation rely on the Goldilocks principle: not too many links, not too few.

A disease has distinct genetic and molecular hallmarks such as sequence variants that are likely to produce the alternative protein structures accountable for individual responses to drugs and disease development. Thus, to set up customized therapies, the structural influences of amino acids on one another need to be tracked down. Using network-based models and classical analysis of amino acid and atomic packing in protein structures, the influence of first shell neighbors on the structural fate of a position upon mutation, is revisited. Regardless of the type and position in a structure, amino acids satisfy on average over their neighbors, a low and similar number of atomic interactions, the average called the neighborhood watch (Nw). The structural tolerance of a position to mutation depends on the modulation of the composition and/or proximity of neighbors to maintain the same Nw, before and after mutation, at every position. Changes, upon mutation of the number of atomic interactions at the level of individual pairs (wij) are structurally tolerated but influence structural dynamics. Robust, fragile and rescue interactions can be identified with Nw and wij, offering a framework to classify sequence variants according to position-dependent structural changes.