Physical Principles Of Biomolecular Recognition, Self-as

生物分子识别的物理原理，自我

• 批准号: 6991159
• 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/6991159
• 关键词: DNA; X ray crystallography; chemical aggregate; chemical binding; collagen; endoplasmic reticulum; gene mutation; human subject; laboratory mouse; molecular assembly /self assembly; molecular chaperones; molecular shape; nucleic acid sequence; nucleic acid structure; osteogenesis imperfecta; spectrometry

Section on Physical Biochemistry, OD/NICHD conducts experimental and theoretical studies of structure and function of biomolecules with emphasis on molecular mechanisms of pathology in connective tissue disorders. Through collaboration with clinical researchers, we strive to gain better knowledge and develop novel techniques for diagnostics, characterization and treatment of osteogenesis imperfecta (OI) and other diseases. Over the years we reported first direct measurements and established physical nature of forces between collagen molecules. We discovered that both procollagen and collagen are intrinsically unstable at physiological conditions so that cells have to use molecular chaperones to fold procollagen within the Endoplasmic Reticulum (ER). We found that micro-unfolding of most thermally labile regions of collagen triple helix is necessary for proper molecular recognition and fiber formation. In fibers, collagen helices are protected from complete unfolding but they constantly undergo transient local unfolding and refolding giving the fibers their unique combination of elasticity and strength. In the last few years the research focus of our group shifted from fundamental studies of these processes to understanding how different OI mutations affect them. In particular, during the last year we extended our collaboration with BEMB/NICHD scientists on studies of physical and chemical properties of mutant collagens from OI patients. We confirmed posttranslational overmodification of type I collagen in several new BEMB patients who were diagnosed with OI but were found to have no type I collagen mutations. The search for the source of this posttranslational overmodification is presently focused on possible defects in molecular chaperones needed for procollagen folding. We discovered structural changes in type I collagen from one long-term BEMB patient, confirming the presence of a mutation not found before. This work prompted BEMB scientists to start screening for more mutations, which could have been missed in initial sequencing. We continued systematic analysis of physical and chemical properties of collagen from the collection of other long-term BEMB patients whose mutations are known. Chartacterization of over a dozen additional mutant collagens confirmed our previous hypothesis that changes in the triple helix stability caused by substitutions of obligatory Gly residues depend primarily on the position of the mutation within certain domains rather than on the the identity of the substituting residue or its immediate local environment. In collaboration with scientists from University of Pavia, Italy and BEMB/NICHD we continued to study the unusual rescue of lethal OI phenotype and moderation of OI symptoms in homozygous mice with dominant G349C substitution in alpha1(I) chain of type I collagen. We found that tissues from heterozygous animals contain substantially smaller amount of molecules with a single mutant chain than expected from known expression level of the mutant allele. Our study of collagen secretion in cell culture revealed that dermal fibroblasts selectively retain and degrade a significant fraction of molecules containing both mutant and normal alpha1(I) chains, while molecules with both mutant chains clear the secretory pathway at almost normal rate. The observed accumulation of unsecreted molecules is likely to cause additional ER stress in collagen producing cells of heterozygous animals and make these cells less viable. Substantially better secretion of molecules containing only mutant alpha1(I) chains is, therefore, likely to improve the viability of fibroblasts and osteoblasts in homozygous animals, potentially explaining their less severe OI phenotype. Additional experiments designed to verify this hypothesis are currently under way. Another important direction of our research is closely related recognition and assembly reactions involviingg DNA. In particular, we uncovered several common physical principles, which govern formation, structure and physical properties of collagen and DNA aggregates. We suggested mechanisms for counter-ion specificity in DNA condensation, DNA overwinding from 10.5 base pairs per helical turn in solution to 10.0 bp/turn in aggregates, sequence homology recognition in pairing of duplex DNA and several other phenomena. The present focus of these studies is measurement of sequence effects in formation, structure and properties of DNA aggregates. During the last year, we concentrated on attempts to develop a theory necessary for extracting information on mutal azimuthal alignment of adjacent helices from x-ray diffraction patterns of highly oriented DNA samples. Preliminary analysis of x-ray diffraction data revealed significant biaxial correlations between DNA molecules even at 15 to 20 Angstrom surface-to-surface separation between DNA helices, confirming our previous theoretical estimates and validating the assumptions built into our theory of sequence-dependent electrostatic interactions between DNA.

OD/NICHD物理生物化学部分进行生物分子结构和功能的实验和理论研究，重点是结缔组织疾病病理学的分子机制。通过与临床研究人员的合作，我们努力获得更好的知识，并开发新的技术，用于诊断，表征和治疗骨生成障碍（OI）和其他疾病。多年来，我们首次报道了直接测量结果，并确定了胶原蛋白分子之间力的物理性质。我们发现前胶原和胶原在生理条件下本质上是不稳定的，因此细胞必须使用分子伴侣在内质网（ER）内折叠前胶原。我们发现，胶原蛋白三螺旋的最热不稳定区域的微展开对于适当的分子识别和纤维形成是必要的。在纤维中，胶原蛋白螺旋受到保护，不能完全展开，但它们不断地经历短暂的局部展开和重折叠，使纤维具有独特的弹性和强度。在过去的几年里，我们小组的研究重点从这些过程的基础研究转移到了解不同的OI突变如何影响它们。特别是，在过去的一年里，我们扩大了与BEMB/NICHD科学家的合作，研究OI患者突变胶原蛋白的物理和化学性质。我们证实了翻译后过度修饰的I型胶原蛋白在几个新的BEMB患者谁被诊断为OI，但没有发现有I型胶原蛋白突变。寻找这种翻译后过度修饰的来源目前集中在前胶原折叠所需的分子伴侣中可能存在的缺陷上。我们从一名长期BEMB患者中发现了I型胶原蛋白的结构变化，证实了之前未发现的突变的存在。这项工作促使BEMB科学家开始筛选更多的突变，这些突变可能在最初的测序中被遗漏。我们继续系统地分析其他长期BEMB患者的胶原蛋白的物理和化学性质，这些患者的突变是已知的。十几个额外的突变胶原的表征证实了我们以前的假设，即由强制性Gly残基取代引起的三螺旋稳定性的变化主要取决于某些结构域内的突变位置，而不是取代残基的身份或其直接的局部环境。我们与来自意大利帕维亚大学和BEMB/NICHD的科学家合作，继续研究了在I型胶原α 1（I）链中具有显性G349 C取代的纯合子小鼠中致命OI表型的不寻常拯救和OI症状的缓和。我们发现，杂合动物的组织中含有的具有单个突变链的分子数量远少于根据突变等位基因的已知表达水平所预期的数量。我们在细胞培养中胶原分泌的研究表明，真皮成纤维细胞选择性地保留和降解含有突变和正常α 1（I）链的分子的显着部分，而两个突变链的分子清除分泌途径几乎正常的速度。所观察到的未分泌分子的积累可能在杂合动物的胶原蛋白产生细胞中引起额外的ER应激，并使这些细胞活力降低。因此，仅含有突变体α 1（I）链的分子的实质上更好的分泌可能会改善纯合动物中成纤维细胞和成骨细胞的活力，这可能解释了它们不太严重的OI表型。目前正在进行旨在验证这一假设的其他实验。 我们研究的另一个重要方向是与DNA密切相关的识别和组装反应。特别是，我们发现了几个共同的物理原理，这些原理支配胶原蛋白和DNA聚集体的形成、结构和物理性质。我们提出了反离子特异性的机制，在DNA凝聚，DNA过卷从10.5碱基对每螺旋转在溶液中的10.0 bp/转的聚集体，序列同源性识别配对的双链体DNA和其他几个现象。目前，这些研究的重点是测量DNA聚集体的形成，结构和性质的序列效应。在过去的一年里，我们集中精力试图开发一个理论，从高度取向的DNA样品的X射线衍射图案中提取相邻螺旋的相互方位对齐信息。X射线衍射数据的初步分析显示，即使在DNA螺旋之间的15至20埃的表面-表面分离下，DNA分子之间也存在显着的双轴相关性，这证实了我们以前的理论估计，并验证了我们的DNA之间序列依赖性静电相互作用理论中的假设。