Determination of the structure of the FMN-binding protein

为了研究蛋白质的功能，获得其结构信息是必不可少的。核磁共振技术适合于分析溶液状态下蛋白质结合辅基的详细结构。本研究利用核磁共振波谱技术对FMN结合蛋白的高级结构进行了研究，通过对未标记的FMN结合蛋白和标记的FMN和/或标记的FMN结合蛋白的高级结构的分析，明确了FMN结合蛋白的高级结构与已被广泛研究的结合蛋白黄素氧还蛋白的高级结构不同<15><13>。31 Thr-Trp-Asn的区域与某些黄素氧还蛋白的序列完全相同，但并不位于FMN的异咯嗪环附近，尽管这一区域对黄素氧还蛋白与FMN的结合很重要。FMN结合蛋白的肽链上的一些芳香基团由异咯嗪环形成阵列，这可能影响其氧化还原电位和反应活性。在FMN结合蛋白的情况下，FMN的磷酸基团暴露于分子的表面，而它被掩埋并与黄素氧还蛋白的肽链内部的一些原子相互作用。这些结果支持了定点突变所获得的突变体的特征。为了结合FMN，Trp 32残基在其庞大性或芳香性方面是重要的，尽管Thr 31和Asn 33残基不是那么重要。9个突变体在氧化还原电位上没有太大的差异，只是在某些突变体上观察到游离FMN的氧化还原反应。我们现在正在研究结合FMN的最小区域或氧化还原电位或反应性的决定区域。

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.