Hess Kayla A, Rohler Cade K, Boutwell Dalton R, Snyder Jason M, Buchanan Lauren E
Department of Chemistry, Vanderbilt University, 1234 Stevenson Center Lane, Nashville, Tennessee 37235, USA.
J Chem Phys. 2024 Aug 7;161(5). doi: 10.1063/5.0207523.
Vibrational spectroscopy of protein structure often utilizes 13C18O-labeling of backbone carbonyls to further increase structural resolution. However, sidechains such as arginine, aspartate, and glutamate absorb within the same spectral region, complicating the analysis of isotope-labeled peaks. In this study, we report that the waiting time between pump and probe pulses in two-dimensional infrared spectroscopy can be used to suppress sidechain modes in favor of backbone amide I' modes based on differences in vibrational lifetimes. Furthermore, differences in the lifetimes of 13C18O-amide I' modes can aid in the assignment of secondary structure for labeled residues. Using model disordered and β-sheet peptides, it was determined that while β-sheets exhibit a longer lifetime than disordered structures, amide I' modes in both secondary structures exhibit longer lifetimes than sidechain modes. Overall, this work demonstrates that collecting 2D IR data at delayed waiting times, based on differences in vibrational lifetime between modes, can be used to effectively suppress interfering sidechain modes and further identify secondary structures.
蛋白质结构的振动光谱通常利用主链羰基的13C18O标记来进一步提高结构分辨率。然而，精氨酸、天冬氨酸和谷氨酸等侧链在相同光谱区域内有吸收，这使得对同位素标记峰的分析变得复杂。在本研究中，我们报告二维红外光谱中泵浦脉冲和探测脉冲之间的等待时间可用于抑制侧链模式，基于振动寿命的差异，有利于主链酰胺I'模式。此外，13C18O-酰胺I'模式寿命的差异有助于确定标记残基的二级结构。使用模型无序肽和β-折叠肽，确定虽然β-折叠比无序结构表现出更长的寿命，但两种二级结构中的酰胺I'模式都比侧链模式表现出更长的寿命。总体而言，这项工作表明，基于模式之间振动寿命的差异，在延迟等待时间收集二维红外数据可用于有效抑制干扰侧链模式并进一步识别二级结构。
