Physical Principles Of Biomolecular Recognition, Self-as

OD/NICHD物理生物化学部分对生物分子的结构和功能进行实验和理论研究，重点研究结缔组织疾病的分子病理机制。通过与临床研究人员的合作，我们努力获得更好的知识和开发新的技术来诊断，表征和治疗成骨不全症（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作用力的物理性质。我们发现前胶原蛋白和胶原蛋白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）内折叠前胶原蛋白。我们发现，胶原蛋白三螺旋的大多数热不稳定区域的微展开是正确的分子识别和纤维形成所必需的。在纤维中，胶原蛋白螺旋受到保护，不会完全展开，但它们会不断经历短暂的局部展开和再折叠，从而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。特别是在去年，我们扩大了与BEMB/NICHD科学家的合作，研究成骨不全症患者突变胶原的物理和化学特性。我们在几例诊断为成骨不全但未发现I型胶原突变的新BEMB患者中证实了翻译后I型胶原过修饰。对这种翻译后过度修饰的来源的研究目前主要集中在原胶原折叠所需的分子伴侣中可能存在的缺陷。我们在一名长期BEMB患者身上发现了I型胶原蛋白的结构变化，证实了一种以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从其他已知突变的长期BEMB患者收集的胶原蛋白的物理和化学特性。对十几种其他突变胶原的表征证实了我们之前的假设，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突变在某些结构域内的位置，而不是取代残基的身份或其直接的局部环境。通过与意大利帕维亚大学和BEMB/NICHD的科学家合作，我们继续研究了I型胶原α 1(I)链中G349C显性取代的纯合子小鼠对致死性OI表型的不同寻常的拯救和OI症状的调节。我们发现杂合动物的组织中含有的单突变链分子比已知突变等位基因表达水平所期望的要少得多。我们对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了大量含有突变和正常α 1(I)链的分子，而含有突变和正常α 1(I)链的分子几乎以正常的速度清除分泌途径。观察到的未分泌分子的积累可能会在杂合动物的胶原生成细胞中引起额外的内质网应激，并使这些细胞的存活率降低。因此，仅含有突变α 1(I)链的分子分泌明显更好，可能提高纯合子动物成纤维细胞和成骨细胞的活力，这可能解释了它们不那么严重的成骨不全症表型。目前正在进行旨在验证这一假设的其他实验。

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.