Determination of the structure of the FMN-binding protein

为了研究蛋白质的功能，获得有关其结构的信息是必不可少的。核磁共振技术适用于分析蛋白质结合基团在溶液状态下的详细结构。本研究通过核磁共振谱测定FMN结合蛋白的高级结构，研究了FMN结合蛋白的高级结构与其氧化还原电位、解离常数等物理化学性质之间的相互作用，通过分析非标记FMN结合蛋白和^&lt；15&gt；N和/或^&lt；13&gt；C标记的FMN结合蛋白，明确了FMN结合蛋白的高级结构不同于研究较多的FMN结合蛋白。31Thr-Trp-ASN区域与一些黄毒素的序列相同，但并不靠近FMN的异四氧嘧啶环，尽管在黄毒素的情况下与FMN结合是重要的。FMN结合蛋白肽链上的一些芳香族基团由异四氧嘧啶环排列而成，这可能影响氧化还原电位和反应活性。在FMN结合蛋白的情况下，FMN的磷酸基团暴露在分子的表面，而它被埋藏在黄曲霉毒素肽链内的一些原子中并相互作用。这些结果支持定点突变突变体的特性。对于结合FMN，Trp32残基在其体积或芳香性方面是重要的，尽管Thr31和Asn33残基不是那么重要。除部分突变体观察到游离FMN的氧化还原反应外，其余9个突变体在氧化还原电位点上差异不大。我们现在正在研究结合FMN的最小区域，或者氧化还原电势或反应活性的决定区域。

To investgate the function of the protein, it is essential to obtain the information about its structure. NMR technology is suitable to analyze the detail structure of the protein binding prosthetic group at the state of solution. In this study, we investgated the interaction between the protein structure and its physico-chemical characters, such as redox potential or dissociation constant, by determining the higher structure of FMN-binding protein using NMR spectroscopy.We clearified that the higher structure of FMN-binding protein is different from that of flavodoxin, a well-studied protein binding FMN,by analyzing non-labelled, and ^<15>N and/or^<13>C labelled FMN-binding protein. The region of 31Thr-Trp-Asn, which is only identical region to the sequence of some flavodoxins, isn't located close to isoalloxazine ring of FMN,although it is important to binding FMN at the case of flavodoxin. Some aromatic groups of the peptide chain of FMN-binding protein are formed the array from isoalloxazine ring, and it is suggested that it may influence the redox potential and reactivity. The phosphate group of FMN is exposed to the surface of the molecule at the case of FMN-binding protein while it is buried and interacted with some atoms inside of the peptide chain of flavodoxin. These results support the characters of mutants using site-directedmutagenesis. To bind FMN,the Trp32 residue is important at the point of its bulkiness or aromaticness although Thr31 and Asn33 residue is not so important. All 9 mutants are not so much different each other at the point of redox potential except for observation of redox reaction of dissociated FMN at some mutants. We are now studying the minimum region to bind FMN or the determining region of redox potential or reactivity.