Xu Jishou, Bowden Edmond F
Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, USA.
J Am Chem Soc. 2006 May 31;128(21):6813-22. doi: 10.1021/ja054219v.
The contact domain utilized by horse cytochrome c when adsorptively bound to a C(10)COOH self-assembled monolayer (SAM) was delineated using a chemical method based on differential modification of surface amino acids. Horse cytochrome c was adsorbed at low ionic strength (pH 7.0, 4.4 mM potassium phosphate) onto 10 microm diameter gold particles coated with HS(CH(2))(10)COOH SAMs. After in situ modification of lysyl groups by reductive Schiff-base methylation, the protein was desorbed, digested using trypsin, and the peptide mapped using LC/MS. Relative lysyl reactivities were ascertained by comparing the resulting peptide frequencies to control samples of solution cytochrome c modified to the same average extent. The least reactive lysines in adsorbed cytochrome c were found to be 13, 72, 73, 79, and 86-88, consistent with a contact region located up and to the left (Met-80 side) of the solvent-exposed heme edge (conventional front face view). The most reactive lysines were 39, 53, 55, and 60, located on the lower backside. The proposed orientation features a heme tilt angle of approximately 35-40 degrees with respect to the substrate surface normal. Factors that can complicate or distort data interpretation are discussed, and the generality of differential modification relative to existing in situ methods for protein orientation determination is also addressed.
利用基于表面氨基酸差异修饰的化学方法，描绘了马细胞色素c吸附结合到C(10)COOH自组装单分子层（SAM）时所利用的接触区域。在低离子强度（pH 7.0，4.4 mM磷酸钾）条件下，将马细胞色素c吸附到涂有HS(CH(2))(10)COOH SAMs的10微米直径金颗粒上。通过还原席夫碱甲基化对赖氨酸基团进行原位修饰后，将蛋白质解吸，用胰蛋白酶消化，并使用液相色谱/质谱对肽段进行图谱分析。通过将所得肽段频率与修饰至相同平均程度的溶液细胞色素c对照样品进行比较，确定相对赖氨酸反应性。发现吸附的细胞色素c中反应性最低的赖氨酸为13、72、73、79和86 - 88，这与位于溶剂暴露血红素边缘上方和左侧（传统正面视图，Met - 80侧）的接触区域一致。反应性最高的赖氨酸为39、53、55和60，位于下后侧。所提出的取向特征是血红素相对于底物表面法线的倾斜角度约为35 - 40度。讨论了可能使数据解释复杂化或扭曲的数据因素，还探讨了相对于现有的蛋白质取向原位测定方法，差异修饰的通用性。
