Molecular Dynamics And Vibrational Characteristics Of Me

工作概述：我们的研究工作包括两个主要领域：(1)双层脂质对整体膜蛋白结构重组的调节作用；(2)振动拉曼和红外光谱成像技术的仪器开发和应用。(1)我们对表征生物膜中波动的脂质微域的影响的兴趣最近集中在由脂质单或多不饱和sn-2链和饱和sn-1链组装组成的双层基质中的簇形成。这些脂质微聚集体的侧向压缩性特性有效地对整体膜蛋白内发生的诱导构象变化施加调节影响。在研究光谱特异性脂质双分子层时，适当的酰基链氘化可以单独监测每个链段的振动动力学。我们继续利用拉曼和红外光谱技术，结合冷冻淬火方法，研究由高度不饱和脂质和/或各种饱和脂质和适当的整体膜蛋白组成的模型双层重组系统。确定了与每个脂类和每个链系统相关的已建立的有序/无序参数，以及脂质微团簇形成的量化。了解波动膜微团簇的大小和形成，可以检查脂质微域对蛋白质构象重排的影响。具体来说，利用红外光谱技术，我们详细研究了细菌紫质光循环活性和蛋白质整体膜α螺旋构象柔韧性的脂质控制。（2A）通过将步进扫描干涉测量法与最先进的红外敏感二维焦平面阵列探测器相结合，相当重视加强我们的中红外光谱化学成像显微镜技术。高性能数字成像与无创、高分辨率光谱学的集成允许在各种宿主环境中可视化不同化学物质的空间分布。该技术的力量还体现在同时获取每个空间位置的红外光谱。作为红外成像技术在诊断病理学中的应用的一个例子，我们的研究以组织微阵列的形式涉及大量前列腺组织切片，其中检查了对照，前列腺上皮内肿瘤和肿瘤组织的振动光谱特征，获得了有希望的结果。在这种情况下，我们的成像仪器结合了高灵敏度的线性阵列检测，用于快速记录超立方体光谱数据。为了在光谱上阐明前列腺组织中存在的各种组织学特征，开发了非常大的光谱训练集和适当的光谱度量来区分前列腺组织中发生的十种形态实体。这种方法目前正在发展，特别是用于确定前列腺腺癌，并应用于其他组织病理。对于红外成像仪器，在光学、探测器配置和数据采集模式等方面进行了改进。特别是，我们已经实现了一种广义形式的干涉快速扫描红外光谱成像（与干涉仪步进扫描方法不同），可用于任何类型的焦平面阵列探测器。此外，我们是第一个实现时间分辨傅里叶变换红外光谱成像的团队，例如，可以将半衰期在毫秒量级的重复动态过程可视化。本案例中使用的示例涉及特定的聚合物液晶复合材料。（2B）我们的成像方法也扩展到可见光区域，利用CCD检测和适当的液晶可调滤波器进行波长区分，获得反射光谱。这种特殊的非侵入性反射装置已成功地用于临床场所，用于确定镰状细胞病患者的组织氧合，其中我们检查了一氧化氮刺激，抑制和给药的影响。我们的结论是，镰状细胞病患者表现出组织氧合受损，尽管其静息血流量值比健康的非裔美国人高两倍。此外，当药理学上血流量增加7倍时，组织氧合得到改善，但仍远低于健康受试者。这种多功能可见反射成像方法为血管疾病患者提供了一种有用的实时探测方法，为评估疾病严重程度和疾病进展提供了一种新的手段。

Summary of Work: Our research efforts encompassed two general areas: (1) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (2) the instrumental development and applications of vibrational Raman and infrared spectroscopic imaging techniques. (1) Our interest in characterizing the effects of fluctuating lipid microdomains within biomembranes has recently focused on cluster formation within bilayer matrices comprised of lipid mono- or polyunsaturated sn-2 chain and saturated sn-1 chain assemblies. The lateral compressibility properties of these lipid microaggregates are effective in exerting a modulatory influence on induced conformational changes occurring within integral membrane proteins. In studying spectroscopically specific lipid bilayers, appropriate acyl chain deuteration allows the vibrational dynamics of each chain moiety to be monitored separately. We have continued the utilization of both Raman and infrared spectroscopic techniques, in conjunction with freeze-quenching methodologies, toward examining model bilayer recombinant systems comprised of highly unsaturated lipids and/or a variety of saturated lipids and an appropriate integral membrane protein. Established order/disorder parameters pertinent to each lipid class and to each chain system are determined, as well as a quantization of the formation of lipid microclusters. An understanding of the sizes and formation of fluctuating membrane microclusters allows an examination of the effects of lipid microdomains on protein conformational rearrangements. Specifically, using infrared spectroscopic techniques, we examined in detail the lipid control of both the photocycle activity of bacteriorhodopsin and the conformational flexibility of the protein's integral membrane alpha helices. (2A) Considerable emphasis has been placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan interferometry with state-of-the-art infrared sensitive two-dimensional focal plane array detectors. The integration of high performance digital imaging with noninvasive, high resolution optical spectroscopy allows a visualization of the spatial distribution of distinct chemical species in a variety of host environments. The power of the technique is also manifest in the simultaneous acquisition of an infrared spectrum for each spatial location. As an example of the utility of the infrared imaging technique in diagnostic pathology, promising results were obtained from our studies involving large numbers of prostate tissue sections in the form of tissue microarrays in which the vibrational spectral signatures for control, prostatic intraepithelial neoplastic and tumor tissues were examined. In this case, our imaging instrumentation incorporated highly sensitive linear array detection for rapidly recording hypercube spectral data. For spectroscopically elucidating the various histological features present in prostate tissue, extraordinarily large spectral training sets and appropriate spectroscopic metrics were developed for distinguishing between ten morphological entities occurring in prostatic tissue. This approach is currently being developed for ascertaining prostatic adenocarcinoma, in particular, and for application to other tissue pathologies. With regard to the infrared imaging instrumentation, a number of enhancing features were made in the optics, in detector configurations, and in data collection paradigms. In particular, we have implemented a generalized form of interferometric rapid-scan infrared spectroscopic imaging (to be distinguished from interferometer step-scan approaches) for utilization with any type of focal plane array detector. Further, we are the first group to implement time-resolved Fourier-transform infrared spectroscopic imaging which permits, for example, the visualization of repetitive dynamic processes with half lives on the order of milliseconds. The example used in this case involved specific polymer liquid crystal composites. (2B) Our imaging approaches have also been extended to the visible spectral region in which reflectance spectra are obtained using CCD detection and appropriate liquid crystal tunable filters for wavelength discrimination. This particular noninvasive reflectance unit has been used successfully in clinical venues for determining tissue oxygenation in patients with sickle cell disease in which we examined the effects of nitric oxide stimulation, inhibition and administration. We concluded that patients with sickle cell disease exhibit impaired tissue oxygenation despite having resting blood flow values that were two-fold higher than healthy African American subjects. Furthermore, when pharmacologically increasing blood flow by seven-fold, tissue oxygenation was improved, but remained well below healthy subjects. This versatile visible reflectance imaging approach suggests a useful, real time probe of patients with vascular disease that allows a novel means for assessing disease severity and disease progression.