RESUMEN
To enhance the photoconversion performance of biphotochromic moxSAASoti protein, a substitution F97 M was introduced. In addition to enhancing the target properties, this substitution also resulted in the crystallization of the recombinant protein within living HeLa cells (also referred to as in cellulo crystallization). The phenomenon of protein crystallization in living cells is not unique, yet the mechanisms and application of in cellulo crystallization remain significant for further research. However, in cellulo crystallization is atypical for fluorescent proteins and detrimental for their biotechnological application. The objective of this study was to elucidate the underlying mechanisms responsible for the crystallization of moxSAASotiF97Min cellulo. For this purpose, the crystal structure of the green form of biphotochromic protein moxSAASotiF97M was determined at high resolution, which surprisingly has a space group, different from those of parent mSAASotiC21N. The analysis provided allowed to propose a mechanism of new crystal contacts formation, which might be a cause of in cellulo protein crystallization.
Asunto(s)
Cristalización , Humanos , Células HeLa , Cristalografía por Rayos X , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Modelos Moleculares , Sustitución de Aminoácidos , Conformación ProteicaRESUMEN
Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of "mox"-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two-point C105 and C117 mutagenesis, we chose C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant. Since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in mSAASoti mainly affect the pKa values of the red form and off-to-on switching. We demonstrate that the microscopic measure of the observed pKa value is the C-O bond length in the phenyl fragment of the neutral chromophore. According to molecular dynamics simulations with QM/MM potentials, larger C-O bond lengths are found for proteins with larger pKa. This feature can be utilized for prediction of the pKa values of red fluorescent proteins.
Asunto(s)
Colorantes , Cisteína , Cisteína/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Mutagénesis , Oxidación-Reducción , Mutación PuntualRESUMEN
Biphotochromic fluorescent protein SAASoti contains five cysteine residues in its sequence and a V127T point mutation transforms it to the monomeric form, mSAASoti. These cysteine residues are located far from the chromophore and might control its properties only allosterically. The influence of individual, double and triple cysteine substitutions of mSAASoti on fluorescent parameters and phototransformation reactions (irreversible green-to-red photoconversion and reversible photoswitching) is studied. A set of mSAASoti mutant forms (C21N, C117S, C71V, C105V, C175A, C21N/C71V, C21N/C175A, C21N/C71G/C175A) is obtained by site-directed mutagenesis. We demonstrate that the C21N variant exists in a monomeric form up to high concentrations, the C71V substitution accelerates photoconversion to the red form and the C105V variant has the maximum photoswitching rate. All C175A-containing variants demonstrate different photoswitching kinetics and decreased photostability during subsequent switching cycles compared with other considered systems. Classical molecular dynamic simulations reveal that the F177 side chain located in the vicinity of the chromophore is considerably more flexible in the mSAASoti compared with its C175A variant. This might be the explanation of the experimentally observed slowdown the thermal relaxation rate, i.e., trans-cis isomerization of the chromophore in mSAASoti upon C175A substitution.