This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Molecular oxygen is a fundamental requirement for aerobic life, yet oxidative stress damages macromolecules and can be lethal if unchecked. Thus far, allosteric conformational changes that regulate transcription in response to the redox state have been visualized in few, if any cases. The long term goal of the proposed research is to elucidate novel and essential allosteric mechanisms that regulate DNA-binding in response to the intracellular redox state. In Gram-positive bacteria, the redox-sensing repressor (Rex) adopts the elegant approach of distinguishing the reduced and oxidized states of the essential cofactor, NAD. Rex binds the DNA target and represses transcription of respiratory genes in the presence of oxidized NAD+, yet authorizes transcription when reduced NADH levels abnormally rise. This laboratory previously determined the X-ray structure of the Rex/NADH complex. Although the Rex/NADH structure provided one important view of the state of Rex that is unable to recognize DNA, the fundamental mechanism of the allosteric response of Rex to NADH/NAD+ remains unknown. The structure of the Rex/NAD+/DNA complex is needed to understand how Rex rearranges to recognize DNA, and how the slight differences between NAD+/NADH trigger this transition. Towards this goal, Rex/NAD+/DNA cocrystals have been obtained. The presence of both DNA and protein was verified from the A280:A260 ratio of dissolved crystals. Although the crystals diffract beyond 3 & resolution, a 300 & c-axis limits our ability to collect complete, high-resolution data in-house. The structure will be solved by MIR or MAD phasing with brominated-DNA, and additional phase information provided by a single SeMet per monomer. Based on the incompatible shapes of Rex/NADH and DNA, Rex is likely to undergo a large conformational change following NADH/NAD+ exchange. The hypothesized conformational rearrangement of Rex would reveal a new paradigm for transcriptional regulation in response to the minimal differences between NAD+ and NADH.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR012408-11
Application #
7602342
Study Section
Special Emphasis Panel (ZRG1-PC (02))
Project Start
2007-07-01
Project End
2008-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
11
Fiscal Year
2007
Total Cost
$8,189
Indirect Cost
Name
Brookhaven National Laboratory
Department
Type
DUNS #
027579460
City
Upton
State
NY
Country
United States
Zip Code
11973
Sui, Xuewu; Farquhar, Erik R; Hill, Hannah E et al. (2018) Preparation and characterization of metal-substituted carotenoid cleavage oxygenases. J Biol Inorg Chem 23:887-901
Jacques, Benoit; Coinçon, Mathieu; Sygusch, Jurgen (2018) Active site remodeling during the catalytic cycle in metal-dependent fructose-1,6-bisphosphate aldolases. J Biol Chem 293:7737-7753
Fuller, Franklin D; Gul, Sheraz; Chatterjee, Ruchira et al. (2017) Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers. Nat Methods 14:443-449
Wangkanont, Kittikhun; Winton, Valerie J; Forest, Katrina T et al. (2017) Conformational Control of UDP-Galactopyranose Mutase Inhibition. Biochemistry 56:3983-3992
VanderLinden, Ryan T; Hemmis, Casey W; Yao, Tingting et al. (2017) Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism. J Biol Chem 292:9493-9504
Song, Lingshuang; Yang, Lin; Meng, Jie et al. (2017) Thermodynamics of Hydrophobic Amino Acids in Solution: A Combined Experimental-Computational Study. J Phys Chem Lett 8:347-351
Orlova, Natalia; Gerding, Matthew; Ivashkiv, Olha et al. (2017) The replication initiator of the cholera pathogen's second chromosome shows structural similarity to plasmid initiators. Nucleic Acids Res 45:3724-3737
Firestone, Ross S; Cameron, Scott A; Karp, Jerome M et al. (2017) Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase. ACS Chem Biol 12:464-473
Tajima, Nami; Karakas, Erkan; Grant, Timothy et al. (2016) Activation of NMDA receptors and the mechanism of inhibition by ifenprodil. Nature 534:63-8
Ericson, Daniel L; Yin, Xingyu; Scalia, Alexander et al. (2016) Acoustic Methods to Monitor Protein Crystallization and to Detect Protein Crystals in Suspensions of Agarose and Lipidic Cubic Phase. J Lab Autom 21:107-14

Showing the most recent 10 out of 167 publications