AAA+ ATPases convert ATP hydrolysis into mechanical work. It is clear that both human cells and disease causing pathogens use these protein complexes to physically manipulate orther proteins or DNA to dismantle and reassemble membranes or other organelles, to replicate DNA and traverse cell division, to repair damaged proteins, or to regulate gene expression. The structural basis on which these molecular machines convert ATP hydrolysis into mechanical work, however, is not known. Such knowledge is vital, not only to our fundamental understanding of energy coupling in general, but also to providing clues to manipulate these proteins to promote human health. Indeed, many diseases are associated with defects in one or more of the 80 AAA+ ATPases that are encoded in the human genome. A major impediment to delineating the mechanisms has been our inability to probe detailed conformational changes that are related to steps in ATP binding, hydrolysis, and product release. We hypothesize that defects in these ATPases will manifest themselves in the manner by which these molecular machines cycle through different stages of ATP hydrolysis. We propose to use novel ensemble scattering and fluorescence single-molecule methods, which are complementary to each other, to aquire solution-phase structural knowledge both under equilibrium and in a time-dependent way. To this end, we will use the highly tractable NtrC (from Escherichia coli) and NtrC1 (from Aquifex aeolicus) proteins as models. These proteins interact with the bacterial transcriptional factor, sigma-54, to remodel RNA polymerase to initiate transcription.
In Aim I, the conformational changes associated with different stages of catalysis will be identified using small- and wide- angle x-ray scattering (SAXS &WAXS). Defects in structural dynamics that are associated with crucial amino acid substitutions will also be determined using single-molecule spectroscopic approaches.
In Aim II, the nucleotide-dependent conformational changes that are associated with the formation of the activator/sigma-54 complex will be identified using both SAXS/WAXS and small-angle neutron scattering (SANS). This will allow us to define the functional roles of nucleotide-dependent conformational changes in these molecular machines. In the course of performing this research, new tools will be developed that are expected to be broadly applicable to similar studies of other proteins that are vital for human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069937-04
Application #
7681461
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Jones, Warren
Project Start
2006-09-01
Project End
2011-08-31
Budget Start
2009-09-01
Budget End
2011-08-31
Support Year
4
Fiscal Year
2009
Total Cost
$315,352
Indirect Cost
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Hanson, Jeffrey A; Brokaw, Jason; Hayden, Carl C et al. (2012) Structural distributions from single-molecule measurements as a tool for molecular mechanics. Chem Phys 396:61-71
Chakraborty, Anirban; Wang, Dongye; Ebright, Yon W et al. (2012) Opening and closing of the bacterial RNA polymerase clamp. Science 337:591-5
Flynn, E Megan; Hanson, Jeffrey A; Alber, Tom et al. (2010) Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain. J Am Chem Soc 132:4772-80
Chen, Baoyu; Sysoeva, Tatyana A; Chowdhury, Saikat et al. (2010) Engagement of arginine finger to ATP triggers large conformational changes in NtrC1 AAA+ ATPase for remodeling bacterial RNA polymerase. Structure 18:1420-30
Tan, Yan-Wen; Hanson, Jeffrey A; Yang, Haw (2009) Direct Mg(2+) binding activates adenylate kinase from Escherichia coli. J Biol Chem 284:3306-13
Burrows, Patricia C; Schumacher, Jörg; Amartey, Samuel et al. (2009) Functional roles of the pre-sensor I insertion sequence in an AAA+ bacterial enhancer binding protein. Mol Microbiol 73:519-33
Burrows, Patricia C; Joly, Nicolas; Cannon, Wendy V et al. (2009) Coupling sigma factor conformation to RNA polymerase reorganisation for DNA melting. J Mol Biol 387:306-19
Burrows, Patricia C; Joly, Nicolas; Nixon, B Tracy et al. (2009) Comparative analysis of activator-Esigma54 complexes formed with nucleotide-metal fluoride analogues. Nucleic Acids Res 37:5138-50
Chen, Baoyu; Sysoeva, Tatyana A; Chowdhury, Saikat et al. (2009) ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli. FEBS J 276:807-15
Hanson, Jeffery A; Yang, Haw (2008) Quantitative evaluation of cross correlation between two finite-length time series with applications to single-molecule FRET. J Phys Chem B 112:13962-70

Showing the most recent 10 out of 14 publications