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. The goal of this project is to test, optimize and develop new NMR pulse programs and methods for studying large proteins and protein-protein complexes on high-field instruments, equipped with cryogenic probes. The challenge of working with large proteins comes from the loss of signal due to the unfavorable relaxation properties of the system, as well as, the difficulty of preparing concentrated protein samples that are stable for extended periods of time. The most common approach taken to alleviate this relaxation problem is to prepare proteins that are fully deuterated, i.e. where all non-exchangeable hydrogens atoms are replaced with deuterium atoms. Although this method does allow for larger proteins to be studied by NMR, replacing all non-exchangeable protons with deuterium also limits the amount of information that can be extracted from a protein. Thus, more recently protein samples have been prepared using alternative deuterium labeling strategies, where some of the protons in the aliphatic side chains are preserved. In particular, a very useful approach is to preserve one of the methyl protons on Leucine, Isoleucine or Valine side chains, thus expanding the type of NMR experiments that can be run and consequently increasing the knowledge obtainable from a given protein sample. At NMRFAM, we are working in collaboration with CESG to assess new methods for preparing deuterated proteins by cell-free expression. At the same time, we are also continuously testing, optimizing and further developing NMR experiments that are used for studying deuterated protein samples. Our goal is to develop experiments that are suited for the given labeling scheme of the protein at study, as well as, to make sure that all possible signal that can be extracted from a difficult protein sample is indeed obtained. This is no trivial task on high-field instruments that are equipped with cryogenic probes, where we often find that the same experiment may require different parameters to work optimally on different spectrometers. Finally, a novel approach to study large systems is to prepare proteins that are fully composed of highly regio- and stereoselectively '2'H, '13'C, '15'N -labeled amino acids. These new type of labeled proteins, which have been named as stereo-array isotope-labeled (SAIL) proteins, have been shown to be exceptionally suitable for NMR structural analyses. At NMRFAM we have been working on the optimization and development of NMR experiments for the studying of SAIL labeled protein, including the application of our fast method for data collection HIFI. For the study of protein complexes, we are devising new methods that, by taking advantage of asymmetric labeling schemes, allow the deconvolution of the spectra for the individual components. We can then observe and obtain information about each component of the complex using a single NMR sample, saving money, measuring time and ensuring that all information is obtained under identical conditions. Furthermore, using the same sample we are also developing cross-saturation experiments that take advantage of the particular labeling scheme to study the protein-protein interface.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR002301-26
Application #
8361188
Study Section
Special Emphasis Panel (ZRG1-BCMB-H (40))
Project Start
2011-03-01
Project End
2012-02-29
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
26
Fiscal Year
2011
Total Cost
$13,194
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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