Engineering a More Thermostable Blue Light Photo Receptor Bacillus subtilis YtvA LOV Domain by a Computer Aided Rational Design Method

Abstract

The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the T m of the flavin mononucleotide based fluorescent protein Bacillus Subtilis YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.

LOV-based reporters for fluorescence imaging

“…For example, DsFbFP, derived from a LOV-coding sequence isolated from the marine a-proteobacterium Dinoroseobacter shibae, exhibits improved fluorescence over PpFbFP [20 ], whilst Crei-LOV, obtained from a photoreceptor protein from the fresh water alga Chlamydomonas reinhardtii, shows enhancements in brightness, photostability and pH tolerance over LOV-based FPs such as iLOV [22]. Thermal stability enhancements in reporter activity have also been reported for CreiLOV, through targeted mutagenesis of EcFbFP [23] and from genome mining for LOV-coding sequences from thermotolerant organisms [8]. Disappointingly, LOV-coding sequences appear to be absent from extremophiles [24], thus limiting the opportunity to exploit this source of genetic diversity.…”

The photophysics of LOV-based fluorescent proteins — new tools for cell biology

“…Their rapid assembly as well as the robustness of the fluorescence signal have rendered LOV-based FPs valuable quantitative reporters for biotechnological approaches [33][34][35][36][37] . To further establish LOV-based FPs as alternative fluorescent proteins, some of their biochemical and photophysical properties, including the fluorescence quantum yield, thermal stability and photobleaching susceptibility have been optimized in different studies by directed evolution approaches and sitedirected mutagenesis 9,10,12,38,39 and were subsequently determined with individual techniques. However, quantitative conclusions from spectrometry, cytometry and microscopy data vitally depend on the accurate characterization of their spectral properties that have been obtained under defined and comparable conditions.…”

Flavin-based fluorescent proteins: emerging paradigms in biological imaging

“…In addition, DNA shuffling was recently employed to engineer iLOV to develop a photostable variant known as phiLOV (Figure 3b) [35]. Moreover, Song et al described a molecular dynamics-guided mutagenesis approach to enhance the thermal stability of EcFbFP by engineering mutations in the dimerization interface (N107Y, M111F, N124Y), thereby increasing the melting temperature by 31 8C (Figure 3c) [36].…”

“…(c) Computational modeling and optimization were utilized to enhance the thermal stability of EcFbFP by nearly 31 8C by engineering mutations N107Y, M111F, and N124Y. Reproduced from [36]. (d) Fluorescence emission of several existing members of the FbFP family including E. coli codon-optimized BsFbFP, generally known as EcFbFP (Ec), the original BsFbFP from B. subtilis (Bs), PpFbFP from P. putida (Pp), a PpFbFP variant derived from sensory box protein 2 also from P. putida, and known as Pp2FbFP (Pp2), and CreiLOV that was identified in Chlamydomonas reinhardtii via genome mining.…”