Physical Principles Of Biomolecular Recognition

生物分子识别的物理原理

• 批准号: 6534881
• 负责人: Sergey Leikin
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/6534881
• 关键词: DNA; X ray crystallography; chemical aggregate; chemical binding; collagen; human tissue; intermolecular interaction; molecular assembly /self assembly; molecular shape; osteogenesis imperfecta; spectrometry

Interactions between various biological helices control protein folding and assembly, DNA packing, protein-DNA interactions, connective tissue formation and stability, and many other processes responsible for normal function and pathology in living organisms. By combining several experimental techniques (UV-VIS, fluorescence, CD and FTIR spectroscopy, x-ray diffraction, calorimetry, etc.) with rigorous physical theories, we continued to advance our understanding of these most basic molecular recognition reactions. Our most significant achievements during the past year were: (1) By combining ultra-slow scanning calorimetry and isothermal circular dichroism we found that at physiological conditions human lung collagen monomers denature within a couple of days and rat tail tendon collagen monomers denature within hours. Contrary to the wide-held belief, the energetically preferred conformation of monomeric collagen at body temperature is a random coil rather than a triple helix. Our data suggest that once secreted from cells collagen helices begin to unfold. Initial micro-unfolding of their least stable domains triggers self-assembly of fibers where the helices are protected from complete unfolding. Apparently, Nature adjusts collagen hydroxyproline content to ensure that the melting temperature of triple helical monomers is several degrees below rather than above body temperature. (2) We further characterized the effect of an insertion of a Gly-Ala-Hyp triplet near C terminal in a1(I) chain in a lethal human OI, an unusual mutation recently discovered by the group of Dr. Marini (HDB/NICHD). By comparing the kinetics of N-propeptide cleavage by type I collagen N-protease, we demonstrated that the insertion leads to a register shift along the whole length of the triple helix rather than "looping out" of the tripeptide. The register shift causes a conformational change in the N-propetide (some 850 residues away from the mutation site), a change in the recognition of the propeptide by N-protease and a change in the specific cleavage kinetics. (3) We determined the extent of posttranslational overmodification of collagen in different tissues from Brtl IV mouse model of osteogenesis imperfecta developed by the group of Dr. Marini. Our present data indicate that the overmodification may not be a significant factor in phenotype variation observed in these mice. (4) We developed a new difference gel electrophoresis technique for traditional protein gels. The technique is based on fluorescent labeling of proteins by different dyes so that differently labeled proteins can be mixed together for further analysis. It is particularly useful for analysis of small differences in the molecular weight (e.g., posttranslational overmodification) and comparison of enzymatic processing of wild type and mutant proteins by co-processing differently labeled proteins in the same test tube. (4) We developed a theory relating the microscopic physics of helix-helix interaction to macroscopic properties of cholesteric DNA assemblies. Estimates based on this theory rationalized the large value of the observed cholesteric pitch and its nonmonotonic dependence on the spacing between DNA molecules.

各种生物螺旋之间的相互作用控制着蛋白质折叠和组装，DNA包装，蛋白质-DNA相互作用，结缔组织的形成和稳定性，以及生物体中正常功能和病理的许多其他过程。通过将几种实验技术（UV-VIS，荧光，CD和FTIR光谱，x射线衍射，量热等）与严格的物理理论相结合，我们继续推进我们对这些最基本的分子识别反应的理解。在过去的一年中，我们最重要的成果是：(1)通过超慢扫描量热法和等温圆二色相结合，我们发现在生理条件下，人肺胶原单体在几天内变性，大鼠尾肌腱胶原单体在几小时内变性。与广泛持有的观点相反，单体胶原蛋白在体温下的能量偏好构象是随机线圈而不是三螺旋。我们的数据表明，一旦从细胞分泌胶原蛋白螺旋开始展开。其最不稳定区域的初始微展开触发纤维的自组装，其中螺旋被保护免受完全展开。显然，自然调节胶原羟基脯氨酸含量，以确保三螺旋单体的熔化温度比体温低几度，而不是高于体温。(2)我们进一步表征了在致死性人OI中a1(I)链C端附近插入Gly-Ala-Hyp三联体的影响，这是Marini博士（HDB/NICHD）小组最近发现的一种不寻常的突变。通过比较I型胶原n蛋白酶切割n -前肽的动力学，我们证明了插入导致沿整个三螺旋长度的寄存器移位，而不是三肽的“环出”。登记移位引起n -肽的构象变化（离突变位点约850个残基），n -蛋白酶对前肽的识别发生变化，并引起特定切割动力学的变化。(3)我们测定了Marini博士小组开发的成骨不全Brtl IV小鼠模型中不同组织中胶原翻译后过修饰的程度。我们目前的数据表明，过度修饰可能不是在这些小鼠中观察到的表型变异的重要因素。(4)对传统蛋白凝胶进行了差异凝胶电泳。该技术基于用不同染料对蛋白质进行荧光标记，这样不同标记的蛋白质就可以混合在一起进行进一步分析。这对于分析分子量的微小差异（例如，翻译后过修饰）以及通过在同一试管中共同处理不同标记的蛋白质来比较野生型和突变型蛋白质的酶处理特别有用。(4)我们发展了一种理论，将螺旋-螺旋相互作用的微观物理与胆甾体DNA组装体的宏观特性联系起来。基于这一理论的估计合理化了所观察到的胆甾体音高的大值及其对DNA分子间距的非单调依赖。