Effects of Proline Substitutions on the Thermostable LOV Domain from Chloroflexus aggregans

Abstract

Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2 °C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family.

Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species

“…In addition, thermal stability measurements of several variants of CagFbFP also revealed two melting temperatures. In prolinesubstituted CagFbFP variants, the temperature of both transitions is raised by up to ~ 2°C [31]. In the variants generated to achieve spectral tuning, both transitions are shifted to lower temperatures [32,33].…”

Tolerance to Glutaraldehyde in Escherichia coli Mediated by Overexpression of the Aldehyde Reductase YqhD by YqhC

“…Substitutions of proline residues to other amino acids are often associated with less rigidity of the protein structure ( Boone et al, 2015 ), which may or may not result in loss of function ( Hardy and Nelson, 2000 ). The inclusion of proline residues is a common strategy to improve proteins’ thermal stability ( Zhou et al, 2010 ; Remeeva et al, 2020 ). Proline substitutions can also change transcription factors’ affinity to different promoters ( Fernandez and Plumbridge, 2019 ).…”

Insights into the mechanisms of LOV domain color tuning from a set of high-resolution X-ray structures

“…CagFbFP can be split for detection of protein-protein interactions 74 . It can be further thermostabilized by replacing Ala95 with a proline 75 for testing even more destabilizing alterations. Finally, CagFbFP can be crystallized and produce high-resolution structures on par with the best resolution structures available for other LOV proteins (1.17 Å for miniSOG 15 and 1.2 Å for iLOV 76 ).…”