Physical Principles Of Biomolecular Recognition

各种生物螺旋之间的相互作用控制着蛋白质的折叠和组装、DNA包装、蛋白质与DNA的相互作用、结缔组织的形成和稳定，以及许多其他负责生物体正常功能和病理的过程。通过结合几种实验技术(UV-Vis、荧光、CD和FTIR光谱、X-射线衍射、量热法等)。通过严格的物理理论，我们继续推进我们对这些最基本的分子识别反应的理解。我们在过去一年中最显着的成就是：(1)结合超慢扫描量热法和等温圆二色谱，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。与普遍认为的相反，单体胶原在体温下的能量首选构象是随机卷曲而不是三螺旋。我们的数据表明，一旦从细胞中分泌出来，胶原螺旋就开始展开。最不稳定结构域的初始微展开触发了纤维的自组装，其中螺旋被保护而不能完全展开。显然，大自然调整了胶原羟脯氨酸的含量，以确保三螺旋单体的熔融温度低于体温几度，而不是高于体温。(2)我们进一步描述了在致死性人类OI链A1(I)链的C末端附近插入Gly-Ala-Hyp三联体的影响，这是Marini博士最近发现的一种不寻常的突变(HDB/NICHD)。通过比较I型胶原N-蛋白酶对N-前肽的裂解动力学，我们证明插入导致了三螺旋全长的注册移位，而不是三肽的“环出”。该寄存器移位导致N-脱氧核糖核酸的构象改变(距突变部位约850个残基)，改变了N-蛋白酶对前肽的识别能力，并改变了特定的切割动力学。(3)我们利用Marini博士建立的Brtl IV小鼠成骨不全模型，测定了不同组织中胶原蛋白翻译后过度修饰的程度。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差异的显著因素。(4)建立了一种适用于传统蛋白质凝胶的差示凝胶电泳法。这项技术是基于不同染料对蛋白质进行荧光标记，以便将不同标记的蛋白质混合在一起进行进一步分析。它特别适用于分析分子量上的微小差异(例如，翻译后过度修饰)，以及通过在同一试管中共处理不同标记的蛋白质来比较野生型和突变蛋白质的酶处理。(4)发展了一种将螺旋-螺旋相互作用的微观物理与胆甾体DNA组装的宏观性质联系起来的理论。基于这一理论的估计使观察到的胆甾相间距的大值及其对DNA分子间距的非单调依赖变得合理。

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.