Proline substitutions effect on the thermostable lov domain from Chloroflexus aggregans with a computational approach

Light-Oxygen-Voltage (LOV) domains are found in plants, bacteria and fungi, they are conserved part of photoreceptors and bind flavins as chromophores and another function of them is to detect blue light. Pririor, LOV domain variants have been developed as fluorescent reporter proteins (also known as flavin-based fluorescent proteins; FbFPs) because their aptitude to fluorescence under anaerobic conditions as well as their fast-folding kinetics, and their small size of ∼12-16 kDa. For real-time quantitative analysis in the biological processes, FbFPs offer optimistic results. Here, CagFbFP is a small thermostable protein presented as its features as LOV domain-containing properties, procured from Chloroflexus aggregates which are thermostable bacterias. It is delivered from histidine kinase. CagFbFP consists of 107 amino acids, its molecular weight is 11.6 kDa. It consists only of the conserved LOV core domain. CagFbFP is a dimer with a molecular weight of ∼25 kDa. The first FbFPs unfolded at moderate temperatures to be used as fluorescence reporters in thermophilic microbes while the desirable feature for fluorescent proteins are increased thermostability. Interestingly, even CagFbFP is the shortest protein amongst the FbFP proteins currently disclosed, still it has phenomenal thermal stability. In this study, we examined if the naturally inherently thermostable protein CagFbFP could advance stabilizing using rational engineering. There are currently different approaches to protein stabilization. Among these approaches we chose to combine the consensus design with proline substitution for this study. Three sites were identified for proline substitution as well as the thermal stability of the obtained variants was determined. When the experimental data obtained from this study are examined, it was observed that the A95P mutant increased the melting temperature by about 2 °C and no change was observed in the A56P and A58P mutations. Later, variants A56P and A95P were crystallized and the effects of the A58P mutation modelled, and it was determined that their structures were preserved compared to the parent protein.