Physical Principles Of Biomolecular Recognition

OD/NICHD物理生物化学组对生物分子的结构和功能进行实验和理论研究，重点是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，并开发新的技术来诊断、表征和治疗成骨不全(OI)和其他疾病。多年来，我们首次报道了胶原蛋白分子之间作用力的直接测量并确定了其物理性质。我们发现，胶原蛋白三螺旋在生理条件下本质上是不稳定的，大多数热不稳定螺旋区域的微展开对于正确的分子识别和纤维形成是必要的。在纤维中，胶原螺旋受到保护，不会完全展开，但它们不断经历短暂的局部展开和再折叠，使纤维具有独特的弹性和强度组合。目前，我们的研究重点正逐渐从这些过程的基础研究转向了解不同的OI突变是如何影响它们的。特别是，在过去的一年里，我们发现前胶原单体在体温下也是热不稳定的。因此，细胞必须使用分子伴侣来折叠内质网(ER)内的前胶原。此外，除了三螺旋N端90个氨基酸突变的OI胶原蛋白外，前胶原蛋白和胶原蛋白的温度和动力学相似。在与HDB/NICHD科学家的合作中，我们确定了这前90个氨基酸构成了胶原蛋白三螺旋的一个重要的折叠结构域。该区域内的OI突变破坏了整个结构域的结构，导致N-前肽的二级结构异常、相互作用和切割。在纤维中加入未切割的分子会产生直径较小的纤维，强度降低，导致早期脊柱侧弯、高度伸展和关节松弛，这些在其他OI患者中不太常见。通过对具有这些和其他OI突变的胶原的物理和化学性质的系统分析，我们还证明了：专性甘氨酸残基的取代(最常见的OI原因)可能会使胶原三螺旋的熔融温度降低5℃，或者实际上对三螺旋的稳定性没有影响，这取决于突变在特定区域内的位置，而取代残基的一致性或其直接的局部环境似乎不那么重要。胶原稳定性降低约5℃或更多会导致突变分子在生理条件下迅速变性，以至于这些分子不能被结合到纤维中。至少在G349C替换的情况下(唯一可以获得小鼠模型的突变)，OI表型与突变的胶原螺旋与其他基质分子的异常相互作用或成骨细胞的异常功能有关，而不是与结构、物理性质的异常或突变的胶原螺旋之间的相互作用有关。我们的初步数据表明，G349C小鼠的成骨细胞功能异常可能是由于内质网应激导致的，即未能通过正常的分泌途径用一个突变链清除胶原螺旋。 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，我们揭示了几个共同的物理原理，这些原理支配着胶原和DNA聚集体的形成、结构和物理性质。我们提出了DNA缩合中的反离子专一性、溶液中DNA从每个螺旋旋转的10.5碱基对过度卷曲到聚集体中的10.0个碱基对/旋转、双链DNA配对中的序列同源性识别以及其他几种现象的机制。目前这些研究的重点是测量DNA聚集体的形成、结构和性质中的序列效应。在去年，我们将该理论扩展到描述由相邻碱基对之间具有序列依赖扭曲的非理想螺旋形成的聚集体。这些计算表明，同源片段在略低浓度的凝聚反离子时应该表现出聚集，并且这种聚集体的结构应该与非同源DNA片段形成的聚集体的结构相区别。我们的预测使之前的几个实验观察结果更加合理，例如，DNA在聚集体中的扭转变形。最重要的是，我们为DNA聚集的可测量参数制定了几个预测，并在目前正在进行的体外实验中进行测试。

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 collagen triple-helix is intrinsically unstable at physiological conditions and that micro-unfolding of most thermally labile helical regions 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. Currently, our research focus is gradually shifting from fundamental studies of these processes to understanding how different OI mutations affect them. In particular, during the last year we found that procollagen monomers are also thermally unstable at body temperature. Therefore, cells have to use molecular chaperones to fold procollagen within Endoplasmic Reticulum (ER). Furthermore, the temperature and kinetics of procollagen and collagen denturation are similar, except for OI collagens with mutations in the N-terminal 90 amino acids of the triple helix. In collaboration with HDB/NICHD scientists, we established that these first 90 amino acids form an important folding domain of collagen triple helix. OI mutations within this region disrupt the structure of the whole domain resulting in abnormal secondary structure, interaction and cleavage of N-propeptides. Incorporation of uncleaved molecules into fibers produces smaller diameter fibers with reduced strength, causing early onset scoliosis, hyper extensibility and joint laxity which are less common in other OI patients. From systematic analysis of physical and chemical properties of collagen with these and other OI mutations, we also demonstrated the following: Substitutions of obligate Gly residues (most common OI cause) may reduce the melting temperature of collagen triple helix by 5 C or have virtually no effect on triple helix stability depending on the position of the mutation within certain domains, while the identity of the substituting residue or its immediate local environment appear to be less important. Reduction of collagen stability by about 5 C or more results in such rapid denaturation of mutant molecules at physiological conditions that these molecules cannot be incorporated into fibers. At least in the case of G349C substitution (the only mutation for which a mouse model is available), OI phenotype is related to abnormal interactions of mutant collagen helices with other matrix molecules or abnormal function of osteoblasts rather than to abnormal structure, physical properties or interactions between mutant collagen helices. Our preliminary data indicate that abnormal osteoblast function in G349C mice may be caused by ER stress resulting from failure to clear collagen helices with one mutant chain through normal secretion pathway. Another important direction of our research is closely related recognition and assembly reactions involving 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 extended the theory to describe formation of aggregates from non-ideal helices with sequence-dependent twist between adjacent base pairs. These calculations showed that homologous fragments should exhibit aggregation at slightly lower concentration of condensing counterions and that the structure of such aggregates should be distinguishable from the structure of aggregates formed by nonhomologous DNA fragments. Our, predictions rationalized several previous experimental observations, e.g., torsional deformation of DNA in aggregates. Most importantly, we formulated several predictions for measurable parameters of DNA aggregation to be tested in in vitro experiments which are presently under way.