Fluorescent proteins contain an internal chromophore constituted of amino acids or an external chromophore covalently bonded to the protein. To increase their fluorescence intensities, many research groups have attempted to mutate amino acids within or near the chromophore. Recently, a new type of fluorescent protein, called UnaG, in which the ligand binds to the protein through many noncovalent interactions was discovered. Later, a series of mutants of the UnaG protein were introduced, which include eUnaG with valine 2 mutated to leucine emitting significantly stronger fluorescence than the wild type and V2T mutant, in which valine 2 is mutated to threonine, emitting weaker fluorescence than the wild type. Interestingly, the single mutation sites of both eUnaG and V2T mutants are distant from the fluorophore, bilirubin, which renders the mechanism of such fluorescence enhancement or reduction unclear. To elucidate the origin of fluorescence intensity changes induced by the single mutations, we carried out extensive analyses on MD simulations for the original UnaG, eUnaG and V2T, and found that the bilirubin ligand bound to eUnaG is conformationally more rigid than the wild-type, particularly in the skeletal dihedral angles, possibly resulting in the increase of quantum yield through a reduction of non-radiative decay. On the other hand, the bilirubin bound to the V2T appears to be flexible than that in the UnaG. Furthermore, examining the structural correlations between the ligand and proteins, we found evidence that the bilirubin ligand is encapsulated in different environments composed of protein residues and water molecules that increase or decrease the stability of the ligand. The changed protein stability affects the mobility and confinement of water molecules captured between bilirubin and the protein. Since the flexible ligand contains multiple hydrogen bond (H-bond) donors and acceptors, the H-bonding structure and dynamics of bound water molecules are highly correlated with the rigidity of the bound ligand. Our results suggest that, to understand the fluorescence properties of protein mutants, especially the ones with noncovalently bound fluorophores with internal rotations, the interaction network among protein residues, ligand, and water molecules within the binding cavity should be investigated rather than focusing on the local structure near the fluorescing moiety. Our in-depth simulation study may offer a foundation for the design principles for engineering this new class of fluorescent proteins.

中文翻译：

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集体非共价相互作用诱导的配体激活的荧光蛋白的荧光增强†

荧光蛋白包含由氨基酸构成的内部发色团或与该蛋白共价结合的外部发色团。为了增加其荧光强度，许多研究小组已尝试使发色团内部或附近的氨基酸突变。最近，发现了一种新型的荧光蛋白，称为UnaG，其中配体通过许多非共价相互作用与该蛋白结合。后来，引入了一系列UnaG蛋白突变体，其中包括缬氨酸2突变为亮氨酸的eUnaG，其荧光强度比野生型强； V2T突变体，其中缬氨酸2突变为苏氨酸，其荧光强度比野生型弱。 。有趣的是，eUnaG和V2T突变体的单个突变位点都与荧光团，胆红素，这使得这种荧光增强或减少的机理不清楚。为了阐明由单个突变引起的荧光强度变化的起源，我们在MD模拟中对原始的UnaG，eUnaG和V2T进行了广泛的分析，发现与eUnaG结合的胆红素配体在结构上比野生型更坚硬，特别是在骨骼的二面角，可能会通过减少非辐射衰变而导致量子产率的提高。另一方面，与V2T结合的胆红素似乎比UnaG中的灵活。此外，检查配体与蛋白质之间的结构相关性，我们发现证据表明胆红素配体被封装在由蛋白质残基和增加或降低配体稳定性的水分子组成的不同环境中。改变后的蛋白质稳定性会影响在胆红素和蛋白质之间捕获的水分子的迁移率和限制。由于柔性配体包含多个氢键（氢键）供体和受体，因此结合的水分子的氢键结构和动力学与结合的配体的刚度高度相关。我们的结果表明，要了解蛋白质突变体的荧光特性，尤其是那些具有非共价结合的具有内部旋转的荧光团的突变体，蛋白质残基之间的相互作用网络，配体，应该研究结合腔内的水分子，而不是集中在发荧光部分附近的局部结构上。我们的深入模拟研究可能为设计此类新型荧光蛋白的设计原理提供基础。