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. Membrane proteins contribute 20 ?30% of all known proteins. However, they present a challenge to the study of their structure and function. X-ray crystallography provides detailed structural information at atomic resolution, but usually membrane proteins has to be crystallized in detergents, which do not faithfully represent the native environment of lipid membranes, destabilize proteins, too much labor is involved in making crystallization attempts, and it is just too difficult or impossible to crystallize certain proteins. Therefore, other methods and approaches are in wide use to study membrane proteins, being in particular true when it comes to the natural lipid environment. Nanodiscs, which are small patches of phospholipid bilayer of controlled size, were developed in the last decade as an alternative to detergents [1, 2] and have been successfully applied to study membrane proteins [3]. Pulsed dipolar spectroscopy (PDS) combined with nitroxide spin-labeling has grown into a highly useful technique to reveal functional mechanisms of lipid-reconstituted membrane proteins [4, 5]. The method is based on measuring distances between cysteine-specific paramagnetic labels introduced into desired position(s) in a protein. Despite its successful application to proteins reconstituted into lipid vesicles, PDS does experiences sensitivity problems due to a relatively small volume of the sample occupied by the protein. This limited volume increases local protein concentration and by the virtue of two-dimensional spatial distribution modifies the signal, making it difficult to isolate the informative part of the signal. In result, longer time for signal averaging is necessary and often moderately long distances cannot be measured. Recently, a PDS study was performed on a bacterial membrane transporter reconstituted into nanodiscs, and large improvement of data quality was reported [6]. Given the fact that ACERT set high priority to the studies on membrane proteins, we consider the development of routine use of lipidic nanodiscs for the studies on structure and function of membrane proteins of utmost importance to ACERT's core research and collaboration. By combining this method with increasing the sensitivity of ACERT PDS instrumentation, we expect very significant improvement of the quality of PDS data and substantial reduction of data averaging time, which are important contributing factors for increasing the efficiency in this direction. [1]. T. H. Bayburt, Y. V. Ginkova, and S. G. Sligar, Nano Lett. (2002), 2, 853-856;[2]. I. G. Denisov, Y. V. Ginkova, A. A. Lazarides, and S. G. Sligar, J. Am. Chem. Soc. (2004), 126, 3477-87;[3]. T. H. Bayburt, Y. V. Ginkova, and S. G. Sligar, Arc. Biochem. Biophys. (2006), 450, 215-222;[4] E. R. Georgieva, T. F. Ramlall, P. P. Borbat, J. H. Freed, D. Eliezer, J. Am. Chem. Soc. (2008), 130, 12856-12857;[5] P. P. Borbat, K. Surendhran, M. Bortolus, P. Zou, J. H. Freed, H. S. Mchaourab, Plos Biol.(2007), 5, 221-2219;[6]. P. Zou and H. S. Mchaourab, Biophys. J.(2010), 6, L18-L20.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
2P41RR016292-11
Application #
8364070
Study Section
Special Emphasis Panel (ZRG1-BCMB-K (40))
Project Start
2011-09-01
Project End
2012-08-31
Budget Start
2011-09-01
Budget End
2012-08-31
Support Year
11
Fiscal Year
2011
Total Cost
$1,910
Indirect Cost
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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