Solution NMR spectroscopy of integral membrane proteins.
Published: 2005
Language: eng
Number of pages: 114
Description: The current studies of PagP highlight the unique contributions that solution NMR can make to membrane protein structural biology.Solution NMR studies of high molecular weight systems are hampered by rapid signal decay. This problem can be partially circumvented by focusing on slowly relaxing nuclei, like backbone amide 15N- 1H or side chain methyl 13C-1H3 groups, while deuterating all other positions. Even though high resolution structure determinations typically characterize every 1H nucleus in a small (&lt;25 kDa) protein, detailed study of the backbone amide positions alone can provide very detailed and diverse information about protein structure and dynamics.PagP transitions between R and T forms in CYFOS-7, a novel detergent that forms small micelles and supports enzymatic activity. The kinetics and thermodynamics of the exchange have been characterized by 1H- 15N NMR spectroscopy, with DeltaH = -10.7 kcal/mol and DeltaS = -37.5 cal/mol&middot;K for the R &rarr; T transition. The R state is flexible and similar to previously determined structures, while the T state is distinguished by a rigidification of the L1 loop and reorganization of the adjacent beta-bulge region.Solution structures of the bacterial outer membrane enzyme PagP (161 residues) in DPC or OG/SDS detergent were determined on the basis of amide  1H-1H NOES, along with backbone dihedral angle restraints and solvent-amide exchange data. Page is an eight-stranded beta-barrel with an N-terminal amphipathic helix. The active site is located inside the barrel at the membrane extracellular boundary.15N T1 and T2 relaxation rates and steady-state 1H-15N NOEs delineate regions of high mobility in PagP, most notably the long extracellular L1 loop that contains many catalytic residues. T1/T2 ratios are particularly sensitive to nanosecond time scale motions dominated by the overall rotation of the protein. Since protein tumbling is typically anisotropic, T1/T 2 ratios also provide orientational information about individual  15N-1H groups with respect to a fixed molecular frame. This is demonstrated by a determination of domain orientations in maltose binding protein (370 residues) bound to beta-cyclodextrin. An anisotropic rotational diffusion tensor could not be determined for PagP due to pervasive nanosecond time scale internal motions.
Notes: Thesis (Ph.D.)--University of Toronto, 2005.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 66-06, Section: B, page: 3116.