Sane S U, Cramer S M, Przybycien T M
Howard P. Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Anal Biochem. 1999 May 1;269(2):255-72. doi: 10.1006/abio.1999.4034.
We have developed a holistic protein structure estimation technique using amide I band Raman spectroscopy. This technique combines the superposition of reference spectra for pure secondary structure elements with simultaneous aromatic, fluorescence, and solvent background subtraction, and is applicable to solution, suspension, and solid protein samples. A key component of this technique was the calculation of the reference spectra for ordered helix, unordered helix, and sheet, turns, and unordered structures from a series of well-characterized reference proteins. We accurately account for the overlap between the amide I and non-amide I regions and allow for different scattering efficiencies for different secondary structures. For hydrated samples, we allowed for the possibility that bound water spectra differ from the bulk water spectra. Our computed reference spectra compare well with previous experimental and theoretical results in the literature. We have demonstrated the use of these reference spectra for the estimation of secondary structures of proteins in solution, suspension, and dry solid forms. The agreement between our structure estimates and the corresponding determinations from X-ray crystallography is good.
我们利用酰胺I带拉曼光谱技术开发了一种整体蛋白质结构估计技术。该技术将纯二级结构元素的参考光谱叠加与同时进行的芳香族、荧光和溶剂背景扣除相结合，适用于溶液、悬浮液和固体蛋白质样品。该技术的一个关键组成部分是从一系列特征明确的参考蛋白质中计算有序螺旋、无序螺旋、片层、转角和无序结构的参考光谱。我们准确地考虑了酰胺I区域和非酰胺I区域之间的重叠，并考虑了不同二级结构的不同散射效率。对于水合样品，我们考虑了结合水光谱与大量水光谱不同的可能性。我们计算的参考光谱与文献中先前的实验和理论结果相当吻合。我们已经证明了使用这些参考光谱来估计溶液、悬浮液和干燥固体形式的蛋白质二级结构。我们的结构估计与X射线晶体学相应测定之间的一致性良好。
