Remeeva Alina, Nazarenko Vera V, Kovalev Kirill, Goncharov Ivan M, Yudenko Anna, Astashkin Roman, Gordeliy Valentin, Gushchin Ivan
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia.
Institut de Biologie Structurale J.-P. Ebel, Université Grenoble Alpes-CEA-CNRS, Grenoble, France.
Proteins. 2021 Mar 27. doi: 10.1002/prot.26078.
Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins.
光氧电压（LOV）结构域是广泛存在的光感受模块，可用于荧光显微镜、光遗传学以及活性氧的可控生成。目前已知的所有LOV结构域的吸收峰都在约440至约450纳米范围内，尚不清楚通过突变是否能使其发生显著位移。在此，我们通过将基于黄素的热稳定荧光蛋白CagFbFP（Gln148）的关键发色团近端谷氨酰胺氨基酸突变为天冬酰胺、天冬氨酸、谷氨酸、组氨酸、赖氨酸和精氨酸，生成了一组LOV结构域变体。所有突变体的吸收光谱均发生蓝移，其中Q148H变体的最大位移为8纳米。虽然CagFbFP及其Q148N/D/E变体对pH不敏感，但Q148H/K/R在酸性pH下会出现适度的红移。为了进一步深入了解，我们测定了所有研究突变体的高分辨率晶体结构，分辨率从Q148D的1.07埃到Q148R的1.63埃。在一些变体中，氨基酸148仍位于黄素附近，而在Q148K、Q148R以及部分Q148D中，蛋白质的C末端解开，残基148的侧链重新定向远离发色团。我们的结果解释了用带电荷氨基酸取代Gln148时颜色未发生位移的原因，并为合理设计基于黄素的颜色位移荧光蛋白铺平了道路。
