Stepanenko Olesya V, Stepanenko Olga V, Kuznetsova Irina M, Shcherbakova Daria M, Verkhusha Vladislav V, Turoverov Konstantin K
Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave., St. Petersburg 194064, Russian.
Department of Biophysics, Peter the Great St. Petersburg Polytechnic University, 29 Polytechnicheskaya st., St. Petersburg 195251, Russian.
Int J Mol Sci. 2017 May 8;18(5):1009. doi: 10.3390/ijms18051009.
Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains.
从细菌光敏色素的PAS（Per-ARNT-Sim重复序列）和GAF（cGMP磷酸二酯酶/腺苷酸环化酶/FhlA转录激活因子）结构域设计的近红外（NIR）荧光蛋白（FPs）通过一个或两个半胱氨酸残基与胆绿素（BV）发色团共价结合。我们研究了BV与一系列源自最近报道的BphP1-FP蛋白的NIR FP变体的相互作用。后者是由细菌光敏色素RpBphP1改造而来，有两个反应性半胱氨酸残基（PAS结构域中的Cys15和GAF结构域中的Cys256），而其突变体在PAS结构域或GAF结构域中含有单个半胱氨酸残基，或者不含半胱氨酸残基。我们在有无变性剂的情况下对BphP1-FP及其突变体进行了生化和光谱表征。我们发现所有BphP1-FP变体都是单体。我们揭示，含有Cys15或Cys256或两者的BphP1-FP变体的光谱特性仅由共价结合的BV发色团决定。因此，这表明在BV与二聚体NIR FPs（如iRFPs）中的单体相互作用中存在单体间变构效应。很可能，在二聚体NIR FPs中除了Cys256残基之外插入Cys15残基，通过促进BV发色团与PAS和GAF结构域的共价交联来影响BV结合。
