Biophysics of Macromolecular Complexes

大分子复合物的生物物理学

• 批准号: 7337585
• 负责人: rodolfo ghirlando
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/7337585
• 关键词: Biophysics; Macromolecular; Complexes

Chromatin structure. We are interested in the biophysical properties and structure of native chromatin fragments. The chicken folate receptor and beta-globin gene loci are ideal for such structural studies in that (i) the region possesses both condensed and transcriptionally active chromatin and (ii) the system has been extensively studied in terms of gene regulation, allowing us to relate the overall chromatin structure to transcription. The condensed chromatin region, spanning 16 kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes, can be released from the cell nucleus with the restriction enzyme HpaII. We have previously analyzed the hydrodynamic properties of this condensed chromatin fragment and showed that it is an extended rod. This provides insights into the structure of heterochromatin, found interspersed within various genomes. The biophysical and biological tools developed in these studies have allowed us to further expand our investigation. Using an erythroid precursor cell line (6C2), we have analyzed a 16 kbp region of the transcriptionally poised beta-globin gene locus and showed that this chromatin fragment also appears to be an extended rod. In order to circumvent some of the problems associated with single nucleotide polymorphisms observed in chicken erythrocytes, we have further dissected the beta-globin gene cluster into a series of five distinct chromatin fragments ranging from 2.1 to 8.0 kbp in size. We are currently comparing the fragments released from 6C2 cells with those released from 10-day old and adult chicken erythrocytes. As the beta-globin genes are transcribed in 10-day old erythrocytes but inactive in adult erythrocytes, these studies will allow us to relate the structures of heterochromatin with those of transcriptionally active and inactive chromatin. Macromolecular assemblies. In collaboration with members of the Laboratory of Molecular Biology, and other laboratories, protein and protein-nucleic acid assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies extend the biochemical and structural investigations and provide important mechanistic information. A case in point is provided by the recently published work on a 17-residue beta-hairpin carried out in collaboration with Dr. William Eaton and other members of the Laboratory of Chemical Physics. The laser temperature jump kinetic data were consistent with the sedimentation equilibrium thermodynamics describing the reversible monomer-trimer self-association. Together these studies provide information on the folding of the beta-hairpin, namely the support for a zipper mechanism in which structure formation is initiated at the turn and propagated by the sequential formation of cross-strand hydrogen bonds and hydrophobic cluster. This result provides important insights into the process of protein folding.

染色质结构。 我们对天然染色质片段的生物物理性质和结构感兴趣。鸡叶酸受体和β-珠蛋白基因位点是理想的结构研究，因为（i）该区域具有浓缩和转录活性的染色质和（ii）该系统已被广泛研究的基因调控，使我们能够将整体染色质结构与转录。凝聚的染色质区域，跨越16 kbp的DNA两侧发育调控叶酸受体和β-珠蛋白基因，可以从细胞核释放的限制酶HpaII。我们先前已经分析了这个浓缩的染色质片段的流体动力学性质，并表明它是一个延伸的杆。这提供了异染色质结构的见解，发现散布在各种基因组中。 在这些研究中开发的生物物理和生物工具使我们能够进一步扩大我们的调查。使用红系前体细胞系（6C 2），我们已经分析了16 kbp的区域的转录平衡β-珠蛋白基因座，并表明，该染色质片段似乎也是一个延长杆。为了规避与在鸡红细胞中观察到的单核苷酸多态性相关的一些问题，我们进一步将β-珠蛋白基因簇解剖成一系列大小从2.1到8.0 kbp的五个不同的染色质片段。我们目前正在比较从6C 2细胞释放的片段与从10日龄和成年鸡红细胞释放的片段。由于β-珠蛋白基因在10天大的红细胞中转录，但在成人红细胞中无活性，这些研究将使我们能够将异染色质的结构与转录活性和无活性染色质的结构联系起来。 大分子组装体。 与分子生物学实验室和其他实验室的成员合作，蛋白质和蛋白质-核酸组装体的特征在于它们的形状，化学计量和相互作用的亲和力。这些研究扩展了生物化学和结构研究，并提供了重要的机理信息。最近发表的关于17个残基的β-发夹的工作提供了一个很好的例子，这项工作是与威廉·伊顿博士和化学物理实验室的其他成员合作进行的。激光温度跃变动力学数据与描述可逆单体-三聚体自缔合的沉降平衡热力学一致。这些研究一起提供了关于β-发夹折叠的信息，即对拉链机制的支持，其中结构形成在转弯处开始并通过交叉链氢键和疏水簇的顺序形成而传播。这一结果为蛋白质折叠过程提供了重要的见解。