Molecular Dynamics And Vibrational Characteristics Of Me

我的分子动力学和振动特性

• 批准号: 7336242
• 负责人: Ira W. Levin
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7336242
• 关键词: Molecular; Dynamics; Vibrational; Characteristics; Me

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 sizes and formation of fluctuating lipid microdomains within biomembranes, using vibrational infrared and Raman spectroscopy, were focused on lipid cluster formation, aggregate size and their modulatory influence on induced conformational changes occurring within integral membrane proteins. In particular, the compressibilities of systems composed of various lipid microdomains were correlated with intramolecular protein rearrangements. Various reconstituted multilamellar and single shell vesicle assemblies were generated as model systems to demonstrate the effects arising from the lateral compressibility properties of these quantified lipid microaggregates. To study spectroscopically specific bilayer lipid chain order/disorder properties within the membrane microdomains, appropriate lipid acyl chain deuteration was required to allow the vibrational dynamics of the chain moieties to be monitored. Binary mixtures of saturated chain phosphatidylcholines were specifically examined. Various spectroscopic splitting patterns of the methylene bending modes allowed a determination of lipid microdomain size in terms of the number of acyl chains constituting a given lipid cluster. The compressibilities of the lipid assemblies were determined both isothermally and adiabatically. An infrared diamond anvil cell was used to measure bilayer isothermal compressibility. Pressures were defined by monitoring the spectra of a pressure transducing material, while volume changes were measured directly. Adiabatic compressibilities of the lipid dispersions were determined by ultrasonic velocimetry in which the thermotropic response to the velocity of sound is measured. In examining binary lipid mixtures, microdomain sizes were found to be functions of the lipid mole fractions constituting the system. Specifically, the lateral compressibilities of the binary systems and integral membrane protein reorganizations were governed by the effective domain sizes defining the assembly. A variety of light scattering studies were also performed on single shell vesicle systems in efforts to correlate size with bilayer microdomain properties as a function of tempeature. (2) Considerable emphasis was placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan and continuous scanning interferometry with state-of-the-art infrared sensitive two-dimensional focal plane array and linear array detectors. The integration of high performance digital imaging with noninvasive, high resolution infrared 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 one example of the utility of the infrared imaging technique in diagnostic pathology, we applied this approach to both supervised and unsupervised (observer independent) prostate histopathology involving large numbers of tissue samples examined in the form of tissue microarrays. This infrared spectroscopic method eliminates the necessity for chemically stained tissue. Further, the high throughput approach inherent in the use of tissue microarrays allows the efficient and effective acquisition of a sample?s vibrational spectral signature for pathologic analyses of biopsied specimens representative of controls, prostatic intraepithelial neoplasia, benign prostatic hyperplasia and adenocarcinoma. We specifically demonstrate the application of automated histologic segementation for a sseries of archival tissue samples; well-defined tests of statistical significance were incorporated. This approach demonstrates that histopathologic changes can now be defined by biochemistry-based, objective spectroscopic criteria that do not necessarily require a pathologist's intervention or interpretation. In these examples, our imaging instrumentation incorporated highly sensitive linear array and focal plane array detection for rapidly recording hypercube spectral data. For spectroscopically elucidating the various histologic features present in prostate tissue, extraordinarily large spectral training sets and appropriate spectroscopic metrics were developed for distinguishing ten morphological entities occurring in this specific tissue. Both the use of the Maximum Gaussian Likelihood Method and, separately, a probabilistic classification model, allowed an objective, automated delineation of the ten histologic categories to be correct to the order of 95-99%. Fine tuning of the tissue segmentation process was developed. Additionally, receiver operating characteristic curves were used to explore relationships between sensitivity and specificity of the high throughput, spectroscopic delineations for distinguishing adenocarcinoma. These procedures are entirely compatible with current tissue processing procedures. With regard to our general 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 and utilized a generalized form of interferometric rapid-, or continuous, scan infrared spectroscopic imaging (to be distinguished from interferometer step-scan approaches) for utilization with any type of focal plane array detector to image nonreversible dynamic events in the order of seconds. 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 tenths of milliseconds. The examples used in this case involved specified polymer dispersed liquid crystalline composites. In contrast to step-scan interferometer approaches to examine molecular dynamics, we demonstrated the ability to monitor the dynamics of multilamellar lipid bilayers using continuously scanning Fourier transform infrared spectroscopic imaging techniques. The spatially and temporally resolved multilamellar images allowed direct and simultaneous determinations of various physical and chemical properties of the assemblies, including the main thermal gel to liquid crystalline phase transition, comparisons of vesicle diffusion rates in both phases and the variation in lipid bilayer packing properties between the inner and outer lamellae defining the vesicle. Since Fourier-transform infrared spectroscopic imaging data sets are extraordinarily large, data processing becomes the limiting step in visualizing sample heterogeneity and temporal profile evolution. We adapted the Gram-Schmidt vector orthogonalization procedure to interferogram space to provide a significant time saving advantage in processing of one to two orders of magnitude in comparison to conventional spectral processing. Instrumentation was developed to combine both vibrational infrared and Raman spectroscopic imaging of a sample; that is, coarse infrared imaging at the 5-7 micron spatial level was first accomplished with Raman submicron spatial imaging them being introduced. Bench to bedside procedures were carried out through the development of visual enhancements of laparoscopic nephrectomies using 3-CCD camera modifications. The newly acquired images affords the surgeon the ability to accurately detect changes in tissue oxygenation dispite limitations to conventional visible light images.

工作总结：我们的研究工作涵盖两个主要领域：（1）双层脂质对完整膜蛋白结构重组的调节作用，以及（2）振动拉曼和红外光谱成像技术的仪器开发和应用。 (1)我们对使用振动红外和拉曼光谱表征生物膜内波动脂质微域的大小和形成的兴趣集中在脂质簇形成、聚集体大小及其对整合膜蛋白内发生的诱导构象变化的调节影响。特别是，由各种脂质微结构域组成的系统的可压缩性与分子内蛋白质重排相关。生成了各种重构的多层和单壳囊泡组件作为模型系统，以证明这些量化的脂质微聚集体的横向压缩特性所产生的影响。为了研究膜微域内光谱特异性双层脂质链有序/无序特性，需要适当的脂质酰基链氘化以监测链部分的振动动力学。特别检查了饱和链磷脂酰胆碱的二元混合物。亚甲基弯曲模式的各种光谱分裂模式允许根据构成给定脂质簇的酰基链的数量来确定脂质微域的大小。脂质组装体的可压缩性是通过等温和绝热测定的。使用红外金刚石砧池来测量双层等温压缩性。通过监测压力转换材料的光谱来定义压力，同时直接测量体积变化。脂质分散体的绝热压缩性通过超声测速法测定，其中测量对声速的热致响应。在检查二元脂质混合物时，发现微域大小是构成系统的脂质摩尔分数的函数。具体来说，二元系统和完整膜蛋白重组的横向可压缩性由定义组装的有效结构域大小控制。还在单壳囊泡系统上进行了各种光散射研究，试图将尺寸与作为温度函数的双层微区特性相关联。 (2) 通过将步进扫描和连续扫描干涉测量与最先进的红外敏感二维焦平面阵列和线性阵列探测器相结合，我们非常重视增强我们的中红外光谱化学成像显微技术。高性能数字成像与非侵入性高分辨率红外光谱的集成可以实现不同化学物质在各种宿主环境中的空间分布的可视化。该技术的强大之处还体现在同时采集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，我们将这种方法应用于监督和非监督（独立于观察者）前列腺组织病理学，涉及以组织微阵列形式检查的大量组织样本。这种红外光谱方法消除了对组织进行化学染色的必要性。此外，使用组织微阵列固有的高通量方法可以高效且有效地采集样品的振动光谱特征，用于对代表对照、前列腺上皮内瘤变、良性前列腺增生和腺癌的活检标本进行病理分析。我们特别展示了自动组织学分割在一系列档案组织样本中的应用；纳入了明确的统计显着性检验。这种方法表明，现在可以通过基于生物化学的客观光谱标准来定义组织病理学变化，而不一定需要病理学家的干预或解释。在这些示例中，我们的成像仪器结合了高灵敏度线性阵列和焦平面阵列检测，用于快速记录超立方体光谱数据。为了通过光谱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该特定组织中出现的十种形态实体。使用最大高斯似然法和单独的概率分类模型，可以客观、自动地描述十个组织学类别，正确率达到 95-99% 左右。开发了组织分割过程的微调。此外，受试者工作特征曲线用于探索高通量光谱描绘的敏感性和特异性之间的关系，以区分腺癌。这些程序与当前的组织处理程序完全兼容。对于我们的通用红外成像仪器，在光学、探测器配置和数据收集范例中进行了许多增强功能。特别是，我们已经实现并利用了一种通用形式的干涉快速或连续扫描红外光谱成像（与干涉仪步进扫描方法不同），可与任何类型的焦平面阵列探测器一起使用，以秒为单位对不可逆动态事件进行成像。此外，我们是第一个实现时间分辨傅立叶变换红外光谱成像的小组，该成像允许半衰期约为十分之一毫秒的重复动态过程的可视化。本例中使用的例子涉及特定的聚合物分散液晶复合材料。与检查分子动力学的步进扫描干涉仪方法相比，我们证明了使用连续扫描傅里叶变换红外光谱成像技术监测多层脂质双层动力学的能力。空间和时间分辨的多层图像允许直接同时确定组件的各种物理和化学性质，包括主要热凝胶到液晶相变、两个相中囊泡扩散速率的比较以及定义囊泡的内层和外层之间的脂质双层堆积特性的变化。由于傅里叶变换红外光谱成像数据集非常大，数据处理成为可视化样品异质性和时间剖面演变的限制步骤。我们将 Gram-Schmidt 矢量正交化过程应用于干涉图空间，与传统的光谱处理相比，在处理过程中提供一到两个数量级的显着节省时间优势。开发了将样品的振动红外成像和拉曼光谱成像结合起来的仪器；即随着拉曼亚微米空间成像的引入，首次实现了5-7微米空间水平的粗红外成像。通过使用 3-CCD 相机改进开发腹腔镜肾切除术的视觉增强功能，可以进行从实验室到床边的手术。新采集的图像使外科医生能够准确检测组织氧合的变化，尽管传统可见光图像存在局限性。