DENSITOMETRIC ANALYSIS OF SYNCHROTRON XRAY FOOTPRINTING AUTORADIOGRAMS

了解蛋白质的折叠动力学在 揭示它们的生物学功能和结构。光谱学 到目前为止使用的方法在表征 蛋白质的瞬变-中间结构，但新的方法 需要调查三级结构的变化。同步加速器x射线 足迹技术使毫秒级的时间分辨结构 核酸构象和蛋白质-核酸的变化 有待研究的络合物。这种方法与质量相结合 光谱测序方法被用来研究折叠 X射线产生细胞色素c·羟基自由基的动力学 水的辐解引起可检测到的氨基酸修饰 细胞色素c在4毫秒内。去卷曲电喷雾 电离质谱图显示了显著的改性结果。 在+16u间隔以上的离子中，未经修饰的 蛋白。对修饰蛋白的胰蛋白酶多肽进行了分离和鉴定 修饰是定量的。尽管串联实验并没有 已经完成，推测以下残留物是 根据我们的多肽结果进行了修改。F10、F36、F46、Y67、Y74、F82、 和M80在暴露50ms后被修饰，而Y97没有 修改。修改范围与 细胞色素c的溶剂可及性残留图。

Understanding the folding kinetics of proteins is vital in revealing their biological functions and structures. Spectroscopic methods used to date have been quite helpful in characterizing transient-intermediate structures of proteins, but new methods to investigate tertiary structure changes are needed. Synchrotron x-ray footprinting techniques enable millisecond time-resolved structural changes in nucleic acid conformation and protein-nucleic acid complexes to be studied. This approach in conjunction with mass spectrometric sequencing methods is being used to study folding kinetics of cytochrome c. Hydroxyl radicals generated by x-ray radiolysis of water induce detectable amino acid modifications of cytochrome c within 4 milliseconds. Deconvoluted electrospray ionization mass spectra reveal that the prominent modification results in ions at +16 u intervals above the molecular weight of unmodified protein. Tryptic peptides of the modified protein were isolated and the modification quantitated. Although tandem experiments have not been completed, it was presumed that the following residues were modified based on our peptide results. F10, F36, F46, Y67, Y74, F82, and M80 were modified after 50 ms of exposure, while Y97 showed no modifications. The modification extents are correlated with the solvent accessibility residue map of cytochrome c.