Modern nuclear magnetic resonance (NMR) spectroscopy continues to be a central technique in the characterization of the structure and dynamics of proteins, nucleic acids and their complexes. Nevertheless, a significant fraction of the proteins that are known through the analysis of the genomic sequence are inaccessible to solution NMR methods. This is because they are too large, either by themselves or because they require association with large assemblies of lipids, and therefore tumble too slowly for optimal NMR performance. This proposal seeks to continue the development of a novel approach to rendering the NMR relaxation properties of large proteins amenable to the comprehensive and efficient application of modern triple resonance and related solution NMR techniques. The basic approach is to simply arrange for the protein molecule to tumble as a much smaller protein. This is achieved by encapsulating the protein in a reverse micelle system and dissolving the entire assembly in a low viscosity fluid. We calculate protein assemblies as large as 100 kDa could be made to tumble with sufficiently short correlation times to allow the full battery of existing triple resonance techniques to be applied, even without benefit of deuteration. The basic approach has been demonstrated with a small model protein, ubiquitin. Using a set of well-characterized proteins, we will determine the conditions necessary to adequately encapsulate large proteins. This method is also potentially applicable to membrane proteins and we will also adapt the technology for this purpose and apply it to several membrane proteins. A number of technical advances will be explored and include the use of cryogenic probe technology and development of a method of partial alignment to allow access to residual dipolar couplings for structural restraints. Should this general strategy prove successful, it would provide a powerful approach to using high-resolution solution NMR techniques to characterize proteins up to 100 kDa in size.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062874-04
Application #
6723752
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Wehrle, Janna P
Project Start
2001-04-01
Project End
2005-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
4
Fiscal Year
2004
Total Cost
$404,432
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Peterson, Ronald W; Nucci, Nathaniel V; Wand, A Joshua (2011) Modification of encapsulation pressure of reverse micelles in liquid ethane. J Magn Reson 212:229-33
Lefebvre, Brian G; Liu, Weixia; Peterson, Ronald W et al. (2005) NMR spectroscopy of proteins encapsulated in a positively charged surfactant. J Magn Reson 175:158-62
Peterson, Ronald W; Lefebvre, Brian G; Wand, A Joshua (2005) High-resolution NMR studies of encapsulated proteins in liquid ethane. J Am Chem Soc 127:10176-7
Peterson, Ronald W; Wand, A Joshua (2005) Self contained high pressure cell, apparatus and procedure for the preparation of encapsulated proteins dissolved in low viscosity fluids for NMR spectroscopy. Rev Sci Instrum 76:1-7
Shi, Zhengshuang; Peterson, Ronald W; Wand, A Joshua (2005) New reverse micelle surfactant systems optimized for high-resolution NMR spectroscopy of encapsulated proteins. Langmuir 21:10632-7
Peterson, Ronald W; Pometun, Maxim S; Shi, Zhengshuang et al. (2005) Novel surfactant mixtures for NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids. Protein Sci 14:2919-21
Peterson, Ronald W; Anbalagan, Karthik; Tommos, Cecilia et al. (2004) Forced folding and structural analysis of metastable proteins. J Am Chem Soc 126:9498-9
Babu, Charles R; Flynn, Peter F; Wand, A Joshua (2003) Preparation, characterization, and NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids. J Biomol NMR 25:313-23
Flynn, Peter F; Milton, Mark J; Babu, Charles R et al. (2002) A simple and effective NMR cell for studies of encapsulated proteins dissolved in low viscosity solvents. J Biomol NMR 23:311-6