Analysis of Nanoconfined Protein Dielectric Signals Using Charged Amino Acid Network Models

Abstract

Protein slow motions involving collective molecular fluctuations on the timescale of microseconds to seconds are difficult to measure and not well understood despite being essential to sustain protein folding and protein function. Broadband dielectric spectroscopy (BDS) is one of the most powerful experimental techniques to monitor, over a broad frequency and temperature range, the molecular dynamics of soft matter through the orientational polarisation of permanent dipole moments that are generated by the chemical structure and morphological organisation of matter. Its typical frequency range goes from 107 Hz down to 10−3 Hz, being thus suitable for investigations on slow motions in proteins. Moreover, BDS has the advantage of providing direct experimental access to molecular fluctuations taking place on different length-scales, from local to cooperative dipolar motions. The unfolding of the cholera toxin B pentamer (CtxB5) after thermal treatment for 3h at 80°C is investigated by BDS under nanoconfined and dehydrated conditions. From the X-ray structure of the toxin pentamer, network-based models are used to infer the toxin dipoles present in the native state and to compute their stability and dielectric properties. Network analyses highlight three domains with distinct dielectric and stability properties that support a model where the toxin unfolds into three conformations after the treatment at 80°C. This novel integrative approach offers some perspective into the investigation of the relation between local perturbations (e.g. mutation, thermal treatment) and larger scale protein conformational changes. It might help ranking protein sequence variants according to their respective scale of dynamics perturbations.