Getahun Zelleka, Huang Cheng-Yen, Wang Ting, De León Brenda, DeGrado William F, Gai Feng
Department of Chemistry, University of Pennsylvania, Philadelphia 19104, USA.
J Am Chem Soc. 2003 Jan 15;125(2):405-11. doi: 10.1021/ja0285262.
It is well-known that the C=N stretching vibration in acetonitrile is sensitive to solvent. Therefore, we proposed in this contribution to use this vibrational mode to report local environment of a particular amino acid in proteins or local environmental changes upon binding or folding. We have studied the solvent-induced frequency shift of two nitrile-derivatized amino acids, which are, AlaCN and PheCN, in H(2)O and tetrahydrofuran (THF), respectively. Here, THF was used to approximate a protein's hydrophobic interior because of its low dielectric constant. As expected, the C=N stretching vibrations of both AlaCN and PheCN shift as much as approximately 10 cm(-1) toward higher frequency when THF was replaced with H2O, indicative of the sensitivity of this vibration to solvation. To further test the utility of nitrile-derivatized amino acids as probes of the environment within a peptide, we have studied the binding between calmodulin (CaM) and a peptide from the CaM binding domain of skeletal muscle myosin light chain kinase (MLCK(579-595)), which contains a single PheCN. MLCK(579-595) binds to CaM in a helical conformation. When the PheCN was substituted on the polar side of the helix, which was partially exposed to water, the C=N stretching vibration is similar to that of PheCN in water. In constrast, when PheCN is introduced at a site that becomes buried in the interior of the protein, the C=N stretch is similar to that of PheCN in THF. Together, these results suggest that the C=N stretching vibration of nitrile-derivatized amino acids can indeed be used as local internal environmental markers, especially for protein conformational studies.
众所周知，乙腈中的C=N伸缩振动对溶剂敏感。因此，我们在本论文中提出利用这种振动模式来报告蛋白质中特定氨基酸的局部环境，或结合或折叠时的局部环境变化。我们分别研究了两种腈基衍生氨基酸AlaCN和PheCN在H₂O和四氢呋喃（THF）中的溶剂诱导频移。在这里，由于THF的低介电常数，它被用来近似蛋白质的疏水内部。正如预期的那样，当用H₂O取代THF时，AlaCN和PheCN的C=N伸缩振动都向高频方向移动了约10 cm⁻¹，这表明该振动对溶剂化敏感。为了进一步测试腈基衍生氨基酸作为肽内环境探针的效用，我们研究了钙调蛋白（CaM）与骨骼肌肌球蛋白轻链激酶（MLCK(579 - 595)）的CaM结合域中的一个肽段之间的结合，该肽段含有一个单一的PheCN。MLCK(579 - 595)以螺旋构象与CaM结合。当PheCN被取代在螺旋的极性侧，该侧部分暴露于水中时，C=N伸缩振动与水中PheCN的相似。相比之下，当PheCN被引入到蛋白质内部被掩埋的位点时，C=N伸缩与THF中PheCN的相似。总之，这些结果表明腈基衍生氨基酸的C=N伸缩振动确实可以用作局部内部环境标记，特别是用于蛋白质构象研究。
