Physical Principles Of Biomolecular Recognition

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、结缔组织形成和稳定性以及许多其他负责生物体正常功能和病理的过程。通过将多种实验技术（UV-VIS、荧光、CD 和 FTIR 光谱、X 射线衍射、量热法等）与严格的物理理论相结合，我们不断加深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重要的成就是：（1）通过结合超慢扫描量热法和等温圆二色性，我们发现在生理条件下，人肺胶原蛋白单体在几天内变性，鼠尾腱胶原蛋白单体在几小时内变性。与普遍的看法相反，单体胶原蛋白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，胶原蛋白螺旋就开始展开。最不稳定区域的初始微展开会触发纤维的自组装，其中螺旋受到保护而不会完全展开。显然，Nature调整胶原蛋白羟脯氨酸含量，以确保三螺旋单体的熔化温度低于而不是高于体温几度。 (2) 我们进一步表征了在致死性人类 OI 中 a1(I) 链 C 末端附近插入 Gly-Ala-Hyp 三联体的影响，这是 Marini 博士小组 (HDB/NICHD) 最近发现的一种不寻常突变。通过比较 I 型胶原 N-蛋白酶切割 N-前肽的动力学，我们证明插入导致沿着三螺旋整个长度的寄存器移位，而不是三肽的“环出”。寄存器移位导致 N-前肽的构象变化（距突变位点约 850 个残基）、N-蛋白酶对前肽的识别发生变化以及特定切割动力学的变化。 (3)我们从Marini博士团队开发的成骨不全症Brtl IV小鼠模型中确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，过度修饰可能不是这些小鼠中观察到的表型变异的重要因素。 (4)我们针对传统蛋白质凝胶开发了一种新的差异凝胶电泳技术。该技术基于不同染料对蛋白质进行荧光标记，以便可以将不同标记的蛋白质混合在一起进行进一步分析。它对于分析分子量的微小差异（例如翻译后过度修饰）以及通过在同一试管中共同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特别有用。 (4) 我们发展了一种将螺旋-螺旋相互作用的微观物理与胆甾型 DNA 组装体的宏观特性联系起来的理论。基于这一理论的估计合理化了观察到的胆甾螺距的大值及其对 DNA 分子间距的非单调依赖性。

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.