COHERENT INFRARED METHODS FOR STRUCTURAL FLUCTUATIONS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们继续发展和完善相干红外光子回波光谱方法，通过它们的振动相关函数来检测肽和蛋白质的结构波动。目前正在评估外差瞬态光栅和三种脉冲光子回波方法来确定振动频率相关函数，以优化信噪比并延长可以检查结构松弛的时间尺度。我们还在开发方法来确定各种各样的二级结构在不同空间位置的结构波动之间的相关性。我们已经在N-H和O-H（以及同位素变体）拉伸模式区域开发了2D-IR和3D-IR方法。酰胺- i和酰胺- a组合四波实验已经完成，并确定了远端N-H和酰胺羰基之间的耦合。已经开发出具有适当带宽和时间分辨率的红外方法来测量(i)基于本征探针的整体蛋白质旋转；（ii）整个发色团相对于蛋白质的运动；（iii）特定IR活性键的取向运动；（iv）这些运动的序参量和时间常数；(v)红外吸收与发色团吸收的相关性；（vi）同一物种（例如氨基酸）的不同红外跃迁之间的相关性；（vii）通过动态烧孔探测酰胺i子结构和（viii）红外光子回波测量。该资源已经证明了一些重要实验的原理，这些实验旨在利用分子振动跃迁来研究分子的重定向，特别是内部旋转。其基本思想是用一个极化红外脉冲激发一个键振动，然后在稍后的时间用另一个极化红外脉冲探测它。初始偶极子在稍后时间对自身的投影的系综平均是键的取向相关函数。为了进一步利用这些技术，我们正在将2D-IR技术与基于激光的t跳方法相结合。我们正在探索产生脉冲的方法，这些脉冲可以直接吸收到溶剂水或D2O的泛音带中。温度跳跃法将应用于诱导与蛋白质和肽的展开/折叠相关的构象变化，并通过我们的二维红外技术进行探测。我们的实验室正在使用这种结合二维IR/T-jump方法研究t跳诱导的绒毛头部折叠动力学。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We continue to develop and refine the methods of coherent infrared photon echo spectroscopy to examine structural fluctuations in peptides and proteins through their vibrational correlation functions. Heterodyned transient gratings and three pulse photon echo methods for determining vibrational frequency correlation functions are now being evaluated to optimize the signal to noise ratio and extend the time scales over which the structural relaxation can be examined. We are also developing methods to determine the correlations between the structure fluctuations at different spatial locations of a wide variety of secondary structures. We have developed 2D-IR and 3D-IR methods in the region of the N-H and O-H (and isotopic variants) stretch modes. Combined amide-I and amide-A four-wave experiments have been accomplished in which couplings between remote N-H and amide carbonyls have been determined. Infrared methods with appropriate band width and time resolution have been developed to measure (i) The overall protein rotation, based on intrinsic probes; (ii) the motion of the chromophore as a whole relative to the protein; (iii) the orientational motion of the particular IR active bond; (iv) both the order parameters and time constants for these motions; (v) correlations between infrared absorption and chromophore absorption; (vi) correlations between different IR transitions of the same species (e.g. amino acid); (vii) probing the amide-I substructure by dynamic hole-burning and (viii) infrared photon echo measurements. The Resource has proved the principle of a number of important experiments aimed at using molecular vibrational transitions to study molecular reorientation, in particular internal rotations. The basic idea is to excite a bond vibration with a polarized IR pulse and probe it with another polarized IR pulse at a later time. The ensemble average of the projection of the initial dipole onto itself at later time is the orientational correlation function of the bond. To further utilize these techniques, we are combining the 2D-IR technology with laser-based T-jump methods. We are exploring methods of generating the pulses that can be directly absorbed into the overtone bands of the solvent water or D2O. The temperature jump method will be applied to induce conformational changes associated with unfolding/folding in proteins and peptides, probed with our 2D IR technique. A study of the T-jump induced folding dynamics of the villian head piece using this combined 2D IR/T-jump approach is under way in our laboratories.