Chromatin structure and architecture. DNA within the cell nucleus is packaged into chromatin and, at present, a variety of models describe the structure of the condensed 30 nm chromatin fiber observed in vitro. However, evidence for a 30 nm chromatin structure in vivo is currently lacking, except in specialized cells such as mature avian erythrocytes in which all of the chromatin is essentially inactive. We are specifically interested in understanding the organization of DNA within condensed chromatin in vivo, as well as the topological constraints imposed on its higher order imposed by organizing proteins such as CTCF and cohesin. Accordingly, 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. Macromolecular assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies form an integral part of current biochemical, structural and physiological investigations as evidenced by recently published work carried out in collaboration with Dr. Clore. E. coli enzyme I represents the first component of the bacterial phosphotransferase system, a pathway coupling the phosphorylation and active transport of sugars across the cell membrane. Enzyme I autophosphorylation by phosphoenolpyruvate (PEP) is the first step of this process and previous studies have shown that alpha-ketoglutarate (aKG) inhibits enzyme I. We have provided a structural basis for enzyme I inhibition by alpha-ketoglutarate and shown that it not only binds to the active site of enzyme I but also acts as a competitive inhibitor for PEP. A characterization of the self-association of enzyme I in the presence of both PEP and aKG was necessary for a determination of the structure and elucidation of this mechanism, which now provides a direct regulatory link between carbon and nitrogen metabolism in E. coli (Venditti et al., ACS Chemical Biology, 2013). Furthermore, in collaboration with colleagues on the NIH campus and at UT Southwestern, we improved upon analytical ultracentrifugation methodology, one of the primary tools used in the studies mentioned above. This methodology leads to more accurate hydrodynamic parameters and accounts for variations previously observed across different instruments. This is not only important for hydrodynamic modeling that routinely requires the highest accuracy, but also in the context of regulatory issues for biologics (Ghirlando et al., Analytical Biochemistry, 2013).

Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2013
Total Cost
$252,480
Indirect Cost
City
State
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Louis, John M; Baber, James L; Ghirlando, Rodolfo et al. (2016) Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles. PLoS One 11:e0160597
Ghirlando, Rodolfo; Mutskova, Radina; Schwartz, Chad (2016) Enrichment and characterization of ferritin for nanomaterial applications. Nanotechnology 27:045102
Schmidt, Thomas; Ghirlando, Rodolfo; Baber, James et al. (2016) Quantitative Resolution of Monomer-Dimer Populations by Inversion Modulated DEER EPR Spectroscopy. Chemphyschem :
Ma, Jia; Metrick, Michael; Ghirlando, Rodolfo et al. (2015) Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium. Biophys J 109:827-37
Inagaki, Sayaka; Ghirlando, Rodolfo; Vishnivetskiy, Sergey A et al. (2015) G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor in Vitro. Biochemistry 54:4320-9
Vrentas, Catherine; Ghirlando, Rodolfo; Keefer, Andrea et al. (2015) Hfqs in Bacillus anthracis: Role of protein sequence variation in the structure and function of proteins in the Hfq family. Protein Sci 24:1808-19
Roche, Julien; Louis, John M; Aniana, Annie et al. (2015) Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding. J Biomol NMR 61:235-48
Lusvarghi, Sabrina; Ghirlando, Rodolfo; Wong, Chi-Huey et al. (2015) Glycopeptide mimetics recapitulate high-mannose-type oligosaccharide binding and function. Angew Chem Int Ed Engl 54:5603-8
Zhao, Huaying; Ghirlando, Rodolfo; Alfonso, Carlos et al. (2015) A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. PLoS One 10:e0126420
Gillette, William K; Esposito, Dominic; Abreu Blanco, Maria et al. (2015) Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions. Sci Rep 5:15916

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