Assessing the structural and dynamical properties of concentrated solutions of the disordered proteins Histatin 5 and its tandem repeat

Sammanfattning: Intrinsically disordered proteins are distinguished by a lack of distinct three-dimensional structure, existing instead as an ensemble of heterogenous structures. In this research, the effect of crowding on these proteins is investigated using a combined approach of experiment and computer simulation, mainly using coarse-grained simulation models to make simulation computationally feasible at the high concentration conditions crowding is displayed.Firstly, the saliva protein Histatin 5 (Hst5) is studied with SAXS, where a selection of coarse-grained models were evaluated using the SAXS data. It was determined that no model could provide adequate simulation-experiment agreement, but a best-performing model could be established. This model predicted moderate change in structure with crowding in the case of Histatin 5.It was postulated the moderate effect of crowding on Histatin 5 was due to its short sequence-length. Thus, the dimer of Hst5 was formed and subjected to investigation by SAXS and computer simulation for crowding effects. The dimer was more challenging to model with a coarse-grained model, and circular dichroism data suggested secondary structures to be present, which a coarse-grained model cannot capture. Atomistic modelling followed, which however did not perform better than the coarse-grained models, showing the importance of further developing these models to represent intrinsically disordered proteins.Atomistic modelling was also performed at high concentrations of Hst5 5, combined with quasi-elastic neutron spectroscopy to elucidate diffusion behaviour at crowded conditions. Diffusion decreased with increasing protein concentration, with temperature effects following Stokes-Einstein beha- viour and increses in salt content to decrease diffusion. Depending on assumptions on the relation between effective- and translational-diffusion, the atomistic model displayed semi-quantitative agreement with experiment.Using neutral polymeric crowders rather than self-crowding showed no impact on structure, as investigated by SAXS. Using DLS did as well not reveal any crowding impact, with the exception of Ficoll®, where Hst5 seemed to modulate Ficoll® self-crowding behaviour in terms of diffusion, decreasing the self-crowding effect. Several coarse-grained models showed similar non-existant effects on structure by crowding, with small deviations from experiment.Benchmarking three coarse-grained models indicate higher degree of finegraining and additional parameters does necessarily follow the intuitive notion of increasing performance, with the most advanced not having as good performance as the two simpler models in terms of predicting radius of gyration.