New Development of Protein Structure Analysis by Vacuum-Ultraviolet Circular Dichroism Spectroscopy

我们在广岛大学同步辐射中心建立了真空紫外圆二色性分光光度计。该分光光度计应用于蛋白质二级结构的分析如下.变性蛋白质的二级结构分析：在各种溶剂条件下，低至172 nm的VUVCD光谱显示了大量的二级结构保留在高铁肌红蛋白、葡萄球菌核酸酶和硫氧还蛋白的酸、冷、热和盐酸胍变性状态下。而三氟乙醇和甲醇诱导的醇变性状态与其他变性状态不同，α-螺旋含量较高（70%）.基于序列的二级结构预测：通过将神经网络方法与从30种蛋白质的低至160 nm的VUVCD数据获得的α-螺旋和3-链的含量和数量相结合，基于序列的二级结构预测的准确度提高至75%。 关于我们 结合目前最好的序列预测算法，VUVCD数据可以将预测准确率提高到80%以上.与脂质相互作用蛋白质的二级结构分析：与磷脂结合后，α-酸性糖蛋白的α-螺旋含量从11%（2段）增加到50%（7段），β-链含量从40%（11段）增加到10%（4段）。这些结构变化主要发生在配体结合位点，表明膜相互作用在蛋白质功能中的重要作用.淀粉样蛋白二级结构分析：β_2-微球蛋白及其两个肽段（#21 - 31，#21 - 29）在175nm处的VUVCD谱显示形成了特征性的β-折叠结构。对淀粉样蛋白的结构细节进行了理论分析，这些结果表明同步辐射VUVCD光谱提供了蛋白质二级结构的新的、详细的信息，极大地扩展了其在蛋白质结构生物学中的应用。少

We constructed the vacuum-ultraviolet circular dichroism (VUVCD) spectrophotometer at Hiroshima University Synchrotron Radiation Center. This spectrophotometer was applied for the secondary-structure analyses of proteins as follows.1. Secondary-structure analysis of denatured proteins: The VUVCD spectra down to 172 nm in various solvent conditions revealed the considerable amount of secondary structures remaining in the acid-, cold-, heat-, and guanidine-hydrochloride-denatured states of metmyoglobin, staphylococcal nuclease, and thioredoxin. However, the alcohol-denatured states induced by trifluoroethanol and methanol involved a large amount (70%) of α-helix, different from other denatured states.2. Sequence-based prediction of secondary structures: The sequence-based prediction of secondary structures was improved up to 75% accuracy by combining the neural network method with the contents and numbers of a-helix and 3-strand obtained from the VUVCD data of 30 proteins down to 160 nm. … More VUVCD data could enhance the predictive accuracy to over 80% when combined with the currently best sequence-predictive algorithms.3. Secondary-structure analysis of protein interacting with lipid: When bound with phospholipid, α-acid-glycoprotein increased the content of α-helix from 11% (2 segments) to 50% (7 segments) and that of β-strand from 40% (11 segments) to 10% (4 segments). These structure changes occurred mainly at the ligand-binding sites, indicating important role of membrane interaction in protein function.4. Secondary-structure analysis of protein in amyloid: The VUVCD spectra of β_2-microglobulin and its two peptide segments (#21-31, #21-29) down to 175 nm revealed the formation of characteristic β-sheet structures. The details of the amyloid structure are under theoretical analysis.These results demonstrate that synchrotron-radiation VUVCD spectroscopy gives new and detailed information on the protein secondary structures, greatly expanding its applicability to protein structural biology. Less