Biophysics of Macromolecular Complexes

染色质结构和构筑。 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-珠蛋白基因座是此类结构研究的理想基因座，因为(I)该区域具有浓缩的和转录活性的染色质，(Ii)该系统在基因调控方面已被广泛研究，使我们能够将整个染色质结构与转录联系起来。组成浓缩的染色质区域跨越15.5kbp的DNA，两侧是发育调节的叶酸受体和β-珠蛋白基因，可以用限制酶HpaII从细胞核中释放出来。我们之前已经分析了这个浓缩的染色质片段的流体力学性质，并表明它是一个延伸杆。这提供了对散布在不同基因组中的异染色质结构的洞察。 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范围。利用红系前体细胞系(6C2)，我们已经证明了用限制性内切酶NheI和XhoI可以从细胞核中释放出16.2kbp的β-珠蛋白基因位点。在这个特殊的细胞系中，这个染色质区域具有转录不活跃的所有特征。我们分析了这个兼性异染色质片段的流体力学性质，表明它也是一个延伸杆。然而，与15.5kbp的结构性浓缩染色质不同，这个16.2kbp的染色质片段包含较小的组蛋白与核酸的比例。因此，观察到的结构上的相似性突出了染色质结构在适应不同DNA连接物长度方面的灵活性。我们还研究了15.5和16.2kBP染色质片段在较高离子强度下的性质，没有发现任何构象突变的证据，表明这些从核中释放的染色质片段不具有最近描述的一些重组结构的更紧密的构象(Ghirlando和Felsenfeld，J.Mol)。Biol.) 我们使用限制性内切酶BamHI进一步将β-珠蛋白基因簇剖析为一系列大小从2.1到8.0KBP的不同的染色质片段。我们发现，沉淀系数与大小的依赖关系与染色质观察到的延伸棒状性质是一致的。我们目前正在开发方案，以比较从6C2细胞获得的β-珠蛋白基因染色质片段与从10日龄和成年鸡红细胞释放的染色质片段。由于β-珠蛋白基因在10日龄的红细胞中转录，但在成年红细胞中不活跃，这些研究将使我们能够将结构性和兼性异染色质的结构与转录活性和非活性染色质的结构联系起来。 大分子组装。 与分子生物学实验室和其他实验室的成员合作，利用流体力学方法对蛋白质和蛋白质-核酸组合体的形状、化学计量和相互作用的亲和力进行了表征。这些研究扩展了生化和结构研究，并提供了重要的机制信息。最近发表的关于完全酵母ESCRT-I异四聚体的工作就是一个恰当的例子，该工作是与詹姆斯·H·赫尔利博士合作进行的。转运所需的内体分选复合体(ESCRT-I)复合体从酵母到人类是保守的，它指导泛素化跨膜蛋白的溶酶体降解，并在人类中指导人类免疫缺陷病毒(HIV)的萌发。该复合体由Vps23、Vps28、Vps37和Mvb12组成，我们发现这些蛋白以高亲和力组装形成一个拉长的、单分散的1：1：1：1复合体。核心复合体的沉积数据与结构数据一致，对扩展的ESCRT-I结构的流体动力学研究允许对完整的ESCRT-I复合体进行建模，并从机理上了解该复合体如何与内膜相互作用。约25纳米的细长形状和尺寸表明ESCRT-I复合体直接参与调节货物募集和膜重构的机械方面(Kostelansky等人，2007年)。

Chromatin structure and architecture. 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 constitutively condensed chromatin region, spanning 15.5 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 shown that a 16.2 Kbp region of the beta-globin gene locus can be released from the nucleus with the restriction enzymes NheI and XhoI. In this particular cell line, this chromatin region possesses all the hallmarks of transcriptional inactivity. We have analyzed the hydrodynamic properties of this facultative heterochromatin fragment and showed that it is also an extended rod. Unlike the 15.5 Kbp constitutively condensed chromatin, however, this 16.2 Kbp chromatin fragment contains a smaller histone protein to nucleic acid ratio. Therefore, the similarity in structure observed highlights the flexibility of the chromatin structure in accommodating different DNA linker lengths. We have also studied the properties of the 15.5 and 16.2 Kbp chromatin fragments at higher ionic strengths and found no evidence of any abrupt conformational change, demonstrating that these chromatin fragments released from the nucleus did not assume the more compact conformations recently described for some reconstituted structures (Ghirlando and Felsenfeld,J. Mol. Biol.). 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 using the restriction enzyme BamHI. We showed that the dependence of the sedimentation coefficient with size is consistent with the extended rod like properties observed for chromatin. We are currently developing protocols to compare beta-globin genes chromatin fragments obtained 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 constitutive and facultative 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 the complete yeast ESCRT-I heterotetramer carried out in collaboration with Dr. James H. Hurley. The endosomal sorting complex required for transport-I (ESCRT-I) complex is conserved from yeast to humans and directs the lysosomal degradation of ubiquitinated transmembrane proteins and, in humans, the budding of the human immunodeficiency virus (HIV). The complex is composed of Vps23, Vps28, Vps37, and Mvb12 and we show that these proteins assemble with high affinity to form an elongated and monodisperse 1:1:1:1 complex. Sedimentation data for the core complex are consistent with the structural data, and hydrodynamic studies on extended ESCRT-I constructs allow for a modeling of the intact ESCRT-I complex and a mechanistic understanding of how the complex interacts with the endosomal membrane. The elongated shape and dimension of approximately 25 nm indicate that the ESCRT-I complex participates directly in regulating the mechanical aspects of cargo recruitment and membrane remodeling (Kostelansky et al., 2007).