UV resonance Raman (UVRR) is a powerful spectroscopic technique for the study of protein conformation and dynamics. Excitation at ~200 nm selectively enhances secondary amide vibrations, which are sensitive to the peptide backbone secondary structure and local environment. Primary amide bands are also resonance enhanced in UVRR spectra and could be used to report on glutamine and asparagine side chains in biophysical studies of proteins and peptides.ud IR absorption, visible Raman, and UVRR were used to investigate the small primary amide molecule propanamide. Dramatic spectral changes in the primary amide vibrations were observed upon aqueous solvation. Aqueous solvation impacts the dielectric and hydrogen bonding environment of the primary amide group resonance structures. This leads to a decrease in C--O and increase in C--N bond order of the primary amide group and therefore alters the resonance enhancement and vibrational frequencies of the primary amide vibrations substantially. Due to this significant response, several primary amide bands can be used as sensitive environmental markers for the glutamine and asparagine side chains.ud Visible Raman and UVRR spectra of L-glutamine and five derivative molecules, D-glutamine, N-Acetyl-L-glutamine, L-glutamine t-butyl ester, Glycyl-L-glutamine, and L-seryl-L-asparagine, were collected and assigned in the 950-1200 cm-1 region. The OCCC dihedral angle of each was determined from X-ray crystal structures. An empirical relationship between the AmIIIP vibrational frequency and the OCCC dihedral angle was observed. This dependence can be explained by hyperconjugation that occurs between the Cbeta--Cgamma sigma orbital and the C=O pi* orbital of the primary amide group. This interaction induces an increase in the Cbeta--Cgamma bond length. As the Cbeta--Cgamma bond length increases, the stretching force constant decreases, downshifting the AmIIIP band. Due to this sensitivity, the AmIIIP can be used as a structural marker diagnostic of the OCCC dihedral angle, such as in the side chains glutamine and asparagine in peptide and protein conformational studies.
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机译:紫外共振拉曼光谱(UVRR)是一种用于研究蛋白质构象和动力学的强大光谱技术。在〜200 nm处激发选择性地增强了二级酰胺振动,该振动对肽骨架二级结构和局部环境敏感。伯酰胺带在UVRR光谱中也共振增强,可用于报告蛋白质和多肽的生物物理研究中的谷氨酰胺和天冬酰胺侧链。 ud红外吸收,可见拉曼和UVRR用于研究小的伯酰胺分子丙酰胺。水溶液溶解后,伯酰胺振动发生剧烈的光谱变化。溶剂化影响伯酰胺基共振结构的介电和氢键结合环境。这导致伯酰胺基团的C–O降低和C–N键序增加,因此会大大改变伯酰胺基团振动的共振增强和振动频率。由于这种显着的响应,几个伯酰胺带可用作谷氨酰胺和天冬酰胺侧链的敏感环境标志物。 ud L-谷氨酰胺和五个衍生分子D-谷氨酰胺,N-乙酰-L的可见拉曼光谱和UVRR光谱收集-谷氨酰胺,L-谷氨酰胺叔丁酯,甘氨酰-L-谷氨酰胺和L-丝氨酰-L-天冬酰胺,并将其分配在950-1200cm-1区域。由X射线晶体结构确定每个的OCCC二面角。观察到AmIIIP振动频率与OCCC二面角之间的经验关系。这种依赖性可以通过发生在伯酰胺基团的Cbeta-Camma sigma轨道和C = O pi *轨道之间的超共轭来解释。这种相互作用导致Cbeta-Cgamma键长度增加。随着Cbeta-Cgamma键长度的增加,拉伸力常数减小,使AmIIIP谱带下移。由于具有这种敏感性,AmIIIP可以用作OCCC二面角的结构标记诊断,例如肽和蛋白质构象研究中的谷氨酰胺和天冬酰胺侧链。
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