Physical Principles of Biomolecular Recognition, Self-Assembly and Regulation

各种生物螺旋之间的相互作用控制蛋白质折叠和组装，DNA包装，蛋白质-DNA相互作用，结缔组织形成和稳定性，以及负责生物体中正常功能和病理学的许多其他过程。通过结合几种实验技术（紫外可见光谱、荧光光谱、红外光谱、X射线衍射、量热法等），在严谨的物理理论指导下，我们继续推进对这些最基本的分子识别反应的理解。过去一年，我们最重要的成就是：（1）我们对成骨不全（OI）小鼠模型的研究表明，（a）尽管I型胶原α 1（I）链中的G349 C取代导致中度至重度（致死性）OI，但该突变本身对肌腱和脱矿骨中的胶原-胶原相互作用、体外原纤维形成绝对没有影响，和蛋白质稳定性。(b)在一些但不是所有突变动物中观察到的相互作用、原纤维形成和蛋白质稳定性的微妙变化似乎与翻译后蛋白质过度修饰的差异有关，而不是与突变蛋白质表达的不同水平有关。(c)过度修饰的程度在很大程度上取决于动物的年龄。(2)我们开始了系统的研究之间的关系的温度依赖性的胶原纤维和蛋白质的热稳定性。我们的初步结果表明，该蛋白质是亚稳态，而不是在生理温度下稳定。似乎解折叠的初始阶段是蛋白质自组装成纤维的触发器，在纤维中蛋白质被保护免于变性。(3)我们开始研究具有导致链寄存器中断的不寻常突变的人I型胶原蛋白，该突变最近由Marini博士的小组（HDB/NICHD）发现。我们描述了这种突变对蛋白质的稳定性和体外原纤维形成的影响。(4)我们发展了一个理论，描述了DNA双链体之间的静电相互作用的碱基对序列的影响。我们发现，这种相互作用可以导致有效的序列识别和配对的50-200 bp的DNA片段。这也许可以解释为什么在细胞中双链体、无核小体DNA的完整片段之间会出现这种令人困惑的局部配对。据文献报道，这种配对先于同源重组，并且它独立于RecA家族蛋白。

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, 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) Our study of a mouse model of osteogenesis Imperfecta (OI) revealed that (a) Although G349C substitution in a1(I) chain of type I collagen results in moderate to severe (lethal) OI, by itself the mutation has absolutely no effect on collagen-collagen interactions in tendons and demineralized bones, on in vitro fibrillogenesis, and on the protein stability. (b) The subtle changes in interactions, fibrillogenesis and protein stability, observed in some but not all mutant animals, appear to be related to difference in posttranslational protein overmodification rather than to different levels of mutant protein expression. (c) The extent of the overmodification depends strongly on the animal age. (2) We started a systematic study of the relationship between temperature dependence of collagen fibrillogenesis and thermal stability of the protein. Our preliminary results indicate that the protein is metastable rather than stable at physiological temperature. It appears that initial stages of unfolding are the trigger for self assembly of the protein into fibers where it is protected from denaturation. (3) We started investigation of human type I collagen with an unusual mutation that leads to chain register disruption, recently discovered by the group of Dr. Marini (HDB/NICHD). We described the effect of this mutation on the stability and in vitro fibrillogenesis of the protein. (4) We developed a theory that describes the effect of base pair sequence on electrostatic interaction between DNA duplexes. We found that this interaction can lead to an effective sequence recognition and pairing of 50-200 bp DNA fragments. This may explain the puzzling observation of such local pairing between intact fragments of duplex, nucleosome-free DNA in cells. It was reported in the literature that such pairing precedes homologous recombination and that it is independent of RecA family proteins.