蛋白质结构分析技术.pptVIP

1. 数据的统一和还原 2. 同晶置换法 single/multiple isomorphous replacement(SIR/MIR) 3. 分子置换法 Molecular replacement (MR) 4. 多波长反常散射法 Anomalous scattering and multiple wavelength anomalous dispersion（MAD） 5. 差值电子密度法 三. 确定位相 Phasing－核心问题 当前31页，共47页，星期一。 1. 电子密度修饰 2. 分辨率 3. 分析电子密度图 4. 全自动化程序发展 四. 电子密度图诠释 Map interpretation 当前32页，共47页，星期一。 1. R因子 2. 限制性最小二乘修正 3. 全自动化软件 4. 结构模型表达 五. 结构模型精化 Refinement 当前33页，共47页，星期一。 第一节 蛋白质和多肽的 氨基酸序列分析 Synopsis: 1. A practical approach to determining amino acid sequence requires a method to remove one amino acid at a time from a polypeptide chain. 2. This is achieved by the use of phenylisothiocyanate（异 硫氰酸苯酯）as the N-terminal labelling reagent, the Edman method. 3. This technique can only deal with chains of 20 to 60 amino acids, and most proteins are much larger. 4. To complete the process, selective hydrolysis of the polypeptide chain cuts very long polypeptides into specific fragments of more manageable size. 当前1页，共47页，星期一。 N端序列分析 Sanger was the first person to determine a protein sequence, for insulin in 1953. The problem: after labelling the N-terminal amino acid, the polypeptide chain must be hydrolysed to isolate and identify the labelled amino acid. Whats needed is a way to remove the first amino acid without having to hydrolyze the whole chain. This was solved by Per Edman, in Sweden in 1956, by using the reagent phenylisothiocyanate to label the N-terminal end of the polypeptide sample. 当前2页，共47页，星期一。 It works by cleaving the N-terminal AA off and leaving the rest of the peptide intact. The Edman method can be repeated on the peptide remaining after the reaction, so this is a very effective method for obtaining AA sequence information. In fact the whole sequence of a peptide containing many AAs can be determined by Edman method. 当前3页，共47页，星期一。 降解反应分三步 偶联 PITC的异硫氰酸酯基与蛋白的自由?－氨基作用，产生苯氨基硫甲酰蛋白质。 裂解 PTC－蛋白质在无水条件下酸裂解，切下反应的那个残基，暴露出下一个自由?－氨基。 转化 ATZ－氨基酸不稳定，在三氟乙酸水溶液条件下，转化成稳定的PTH－氨基