DENSITOMETRIC ANALYSIS OF SYNCHROTRON XRAY FOOTPRINTING AUTORADIOGRAMS

了解蛋白质的折叠动力学对于 揭示它们的生物学功能和结构。 光谱 迄今为止使用的方法在描述 蛋白质的瞬时中间结构，但新的方法， 需要研究三级结构变化。 同步加速器x射线 足迹技术使毫秒时间分辨的结构 核酸构象和蛋白质-核酸的变化 复合物进行研究。 这种方法结合质量 光谱测序方法被用来研究折叠 细胞色素C动力学X射线产生的羟基 水的辐解诱导可检测的氨基酸修饰， 细胞色素c的变化 去卷积电喷雾 电离质谱显示， 在高于未改性的分子量+16 u间隔的离子中， 蛋白 分离修饰蛋白的胰蛋白酶肽， 修改量化。 尽管串联实验还没有 完成后，假定以下残留物 根据我们的肽结果进行了修改。 F10、F36、F46、Y67、Y74、F82、 和M80在暴露50 ms后发生了改变，而Y 97则没有发生改变。 修改. 修改程度与 细胞色素c溶剂可及性残基图

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.