Chromatin structure and architecture. DNA within the cell nucleus is packaged into chromatin and a variety of models currently describe the structure of the condensed 30 nm chromatin fiber observed in vitro. However, evidence for this structure in vivo is lacking, except in specialized cells such as mature avian erythrocytes in which all of the chromatin is essentially inactive. We are interested in understanding the organization of DNA within condensed chromatin in vivo, as well as the topological constraints imposed on its higher order by organizing proteins such as CTCF and cohesin. We are developing high resolution chromosome capture conformation assays utilizing native chromatin fragments, such as the previously studied condensed heterochromatin flanked by the developmentally regulated folate receptor and beta-globin genes. These studies will allow us to better understand the structure of the chromatin fiber in vivo, thus providing insight in the relations between chromatin structure and essential processes such as gene expression and DNA replication. Macromolecular assemblies of biological interest. Biological assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies complement current investigations, as evidenced by recent work carried out with the laboratory of Dr. Clore. GAG is the primary polyprotein involved in the assembly of the human HIV-1 retrovirus. It is expressed in the cytoplasm of the host cell and transported to the plasma membrane. In the course of the budding process, HIV-1 protease cleaves GAG, leading to the formation of the mature virion. Cleavage of the GAG polyprotein results in the formation of its constituent proteins, namely matrix, capsid, nucleocapsid and the intrinsically disordered p6. Matrix regulates the binding of GAG to the cell membrane and capsid assembles to form the viral capsid. Nucleocapsid binds the viral nucleic acids and p6 interacts with cellular and viral proteins. Structural, biochemical and biophysical studies on GAG and its components are expected to provide important insight into the mechanism of HIV-1 viral assembly and subsequent budding. Initial studies focused on the structure and dynamics of the full-length capsid protein, observed in the form of exchanging monomers and dimers. In the dimer form, the C-terminal domains responsible for dimerization adopt a single orientation. The relative orientations of the N- and C-terminal domains occupy a broad distribution of states that differ significantly for the monomer and dimer forms of the protein. Importantly, the orientations observed for this protein within the HIV-1 capsid assembly are only present in a small subpopulation of the dimer distribution of states (Deshmukh et al., Journal of the American Chemical Society, 2013). Subsequent studies considered the longer capsid-spacer peptide 1-nucleocapsid fragment of GAG. These studies demonstrate that both the capsid and the nucleocapsid retain their individual structure and tumble semi-independently of each other. The addition of nucleic acids, which bind to the nucleocapsid domain and fix the orientation of the two zinc knuckles, does not influence the structure of the capsid domain. However, access of the HIV-1 protease to the spacer peptide 1 nucleocapsid site is enhanced significantly, even though the flexible spacer peptide 1 remains unstructured in the presence of nucleic acids (Deshmukh et al., Angewandte Chemie International Edition English, 2014). Analytical ultracentrifugation is one of the primary tools used for the above mentioned hydrodynamic studies. In collaboration with colleagues from the NIH, and others, we have further improved on the methodology for data collection. This ultimately leads to more accurate hydrodynamic parameters, important in particular for hydrodynamic modeling that routinely requires the highest accuracy (Ghirlando et al., Analytical Biochemistry, 2014; Zhao et al., Analytical Biochemistry, 2014).

Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2015
Total Cost
Indirect Cost
Name
U.S. National Inst Diabetes/Digst/Kidney
Department
Type
DUNS #
City
State
Country
Zip Code
Chiliveri, Sai Chaitanya; Louis, John M; Ghirlando, Rodolfo et al. (2018) Tilted, Uninterrupted, Monomeric HIV-1 gp41 Transmembrane Helix from Residual Dipolar Couplings. J Am Chem Soc 140:34-37
Zhou, Bing-Rui; Jiang, Jiansheng; Ghirlando, Rodolfo et al. (2018) Revisit of Reconstituted 30-nm Nucleosome Arrays Reveals an Ensemble of Dynamic Structures. J Mol Biol 430:3093-3110
Nguyen, Trang T; Ghirlando, Rodolfo; Venditti, Vincenzo (2018) The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by ?-ketoglutarate. J Biol Chem 293:2631-2639
Chittori, Sagar; Hong, Jingjun; Saunders, Hayden et al. (2018) Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N. Science 359:339-343
Lusvarghi, Sabrina; Ghirlando, Rodolfo; Davison, Jack R et al. (2018) Chemical and Biophysical Approaches for Complete Characterization of Lectin-Carbohydrate Interactions. Methods Enzymol 598:3-35
Kang, Hyeog; Oka, Shinichi; Lee, Duck-Yeon et al. (2017) Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins. Nat Commun 8:15560
Xiao, Hua; Wang, Feng; Wisniewski, Jan et al. (2017) Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres. Genes Dev 31:1958-1972
Passos, Dario Oliveira; Li, Min; Yang, Renbin et al. (2017) Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 355:89-92
Jha, Jyoti K; Li, Mi; Ghirlando, Rodolfo et al. (2017) The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2. MBio 8:
Libich, David S; Tugarinov, Vitali; Ghirlando, Rodolfo et al. (2017) Confinement and Stabilization of Fyn SH3 Folding Intermediate Mimetics within the Cavity of the Chaperonin GroEL Demonstrated by Relaxation-Based NMR. Biochemistry 56:903-906

Showing the most recent 10 out of 77 publications