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

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. View Full-Text

Keywords: fluorescent proteins; light-oxygen-voltage (LOV) domain; protein design; thermal stabilization; proline substitution; consensus design; X-ray crystallography

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Remeeva, A.; Nazarenko, V.V.; Goncharov, I.M.; Yudenko, A.; Smolentseva, A.; Semenov, O.; Kovalev, K.; Gülbahar, C.; Schwaneberg, U.; Davari, M.D.; Gordeliy, V.; Gushchin, I. Effects of Proline Substitutions on the Thermostable LOV Domain from Chloroflexus aggregans. Crystals 2020, 10, 256. https://doi.org/10.3390/cryst10040256

AMA Style

Remeeva A, Nazarenko VV, Goncharov IM, Yudenko A, Smolentseva A, Semenov O, Kovalev K, Gülbahar C, Schwaneberg U, Davari MD, Gordeliy V, Gushchin I. Effects of Proline Substitutions on the Thermostable LOV Domain from Chloroflexus aggregans. Crystals. 2020; 10(4):256. https://doi.org/10.3390/cryst10040256

Chicago/Turabian Style

Remeeva, Alina, Vera V. Nazarenko, Ivan M. Goncharov, Anna Yudenko, Anastasia Smolentseva, Oleg Semenov, Kirill Kovalev, Cansu Gülbahar, Ulrich Schwaneberg, Mehdi D. Davari, Valentin Gordeliy, and Ivan Gushchin. 2020. "Effects of Proline Substitutions on the Thermostable LOV Domain from Chloroflexus aggregans" Crystals 10, no. 4: 256. https://doi.org/10.3390/cryst10040256

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

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