This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our laboratory, which is part of the Membrane Protein Structural Biology Consortium of the NIH Protein Structure Initiative, is actively developing technologies to overcome significant bottlenecks in the structure determination of eukaryotic transmembrane proteins. This includes the optimization of solubilization and crystallization conditions to improve the diffraction quality of crystals. We are using SANS (small angle neutron scattering) with contrast variation experiments to determine the spatial distribution of detergent bound to a purified membrane protein, Bor1p, a borate transporter from S. cerevisiae and member of the SLC4 superfamily, an important group of membrane proteins that includes the human anion transporters, AE1, 2, and 3. Because of the inherently different scattering length densities of the detergents and protein it will be possible to determine how the Bor1p/detergent distribution is affected by changes in solution conditions, including changes in detergent type and concentration, and the presence of inhibitors and substrates bound to Bor1p. It is of particular importance to study how this distribution changes depending on the detergent used and how such changes correlate with hydrodynamic and functional properties of the protein. This information can be used to develop strategies for optimizing the choice of detergent conditions that will promote protein crystallization, a process that currently is performed by trial and error. In our initial SANS studies we have found that there is considerable variation in the radius of gyration (Rg) of identical protein purifications of Bor1p, which may result from small differences in the detergent content of the preparations. Prior to our next SANS run at Oak Ridge Laboratories in December 2010, we propose to use SAXS at CHESS to help in identifying the source of the Rg variation through analysis of Bor1p in solution with small variations in detergent concentration. SAXS provides a rapid method of evaluating samples not possible with SANS. The SAXS experiments will be performed on a series of Bor1p/detergent solutions with differing relative amounts of detergent and with the appropriate no protein and no detergent buffer controls. Additionally, we are interested in evaluating SAXS for studying solution conformational changes of Bor1p bound to different protein additives. The results from these experiments will be compared to a set of Bor1p Tm (melting temperatures) measurements that evaluated the effect of different additives on Bor1p Tm.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-BCMB-E (40))
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Cornell University
Schools of Arts and Sciences
United States
Zip Code
Bauman, Joseph D; Harrison, Jerry Joe E K; Arnold, Eddy (2016) Rapid experimental SAD phasing and hot-spot identification with halogenated fragments. IUCrJ 3:51-60
Xu, Caishuang; Kozlov, Guennadi; Wong, Kathy et al. (2016) Crystal Structure of the Salmonella Typhimurium Effector GtgE. PLoS One 11:e0166643
Cogliati, Massimo; Zani, Alberto; Rickerts, Volker et al. (2016) Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population. Fungal Genet Biol 87:22-9
Oot, Rebecca A; Kane, Patricia M; Berry, Edward A et al. (2016) Crystal structure of yeast V1-ATPase in the autoinhibited state. EMBO J 35:1694-706
Lucido, Michael J; Orlando, Benjamin J; Vecchio, Alex J et al. (2016) Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. Biochemistry 55:1226-38
Gupta, Kushol; Martin, Renee; Sharp, Robert et al. (2015) Oligomeric Properties of Survival Motor Neuron·Gemin2 Complexes. J Biol Chem 290:20185-99
Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R et al. (2015) Comparison of Saccharomyces cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site. Structure 23:352-63
Orlando, Benjamin J; Lucido, Michael J; Malkowski, Michael G (2015) The structure of ibuprofen bound to cyclooxygenase-2. J Struct Biol 189:62-6
Wong, Kathy; Kozlov, Guennadi; Zhang, Yinglu et al. (2015) Structure of the Legionella Effector, lpg1496, Suggests a Role in Nucleotide Metabolism. J Biol Chem 290:24727-37
Muñoz-Escobar, Juliana; Matta-Camacho, Edna; Kozlov, Guennadi et al. (2015) The MLLE domain of the ubiquitin ligase UBR5 binds to its catalytic domain to regulate substrate binding. J Biol Chem 290:22841-50

Showing the most recent 10 out of 368 publications