Abstract
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.
Highlights
Phototransformable fluorescent proteins (PTFPs) are essential components when performing super-resolution techniques[1,2,3]
Its biphotochromic nature can be observed on the wild type gene, while other biphotochromic proteins ( IrisFP22, NijiFP23, etc.) were obtained by site-directed mutagenesis of amino acid residues in the chromophore microenvironment (M159A, F173S) in contrast to SAASoti, which does not require these mutations and has a biphotochromic nature
By applying molecular dynamics simulations with the QM/MM potentials of the red forms of C21N and mSAASoti variants we estimated that higher pKa value of the red form in the case of C21N SAASoti is associated with the mean C-O length shortening in the phenyl fragment of the chromophore
Summary
Phototransformable fluorescent proteins (PTFPs) are essential components when performing super-resolution techniques[1,2,3]. In many cases a Cys-containing tripeptide glutathione forms a redox couple of reduced and oxidized form that is one of the key parameters in the living cell. It is known for some microorganisms, e.g., E. coli, glutathionylation of methionine synthase[6] and PAPS reductase[7] can occur during oxidative stress, resulting in inactivation of both enzymes. Other hand, cysteine-free variants of several fluorescent proteins often demonstrate significant photostability loss and decreased b rightness[5,11,12] It is S146C substitution in the chromophore microenvironment of the mKate FP that resulted in the 12-fold more photostable variant named mStable due to sulfoxidation under illumination[13]. The authors[4] suggest using superfolder GFP variant (sfGFP) coupled with an optimized extravasation signal instead of cysteine substitution, as the protein folding rate in this case exceeds the rate of undesirable bonds formation between the proteins
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