A large sample volume magic angle spinning nuclear magnetic resonance probe for in situ investigations with constant flow of reactants.

A large-sample-volume constant-flow magic angle sample spinning (CF-MAS) NMR probe is reported for in situ studies of the reaction dynamics, stable intermediates/transition states, and mechanisms of catalytic reactions. In our approach, the reactants are introduced into the catalyst bed using a fixed tube at one end of the MAS rotor while a second fixed tube, linked to a vacuum pump, is attached at the other end of the rotor. The pressure difference between both ends of the catalyst bed inside the sample cell space forces the reactants flowing through the catalyst bed, which improves the diffusion of the reactants and products.

Comparative dynamics of leucine methyl groups in FMOC-leucine and in a protein hydrophobic core probed by solid-state deuteron nuclear magnetic resonance over 7-324 K temperature range.

Quantitative dynamics of methyl groups in 9-fluorenylmethyloxycarbonyl-leucine (FMOC-leu) have been analyzed and compared with earlier studies of methyl dynamics in chicken villin headpiece subdomain protein (HP36) labeled at L69, a key hydrophobic core position. A combination of deuteron solid-state nuclear magnetic resonance experiments over the temperature range of 7-324 K and computational modeling indicated that while the two compounds show the same modes of motions, there are marked differences in the best-fit parameters of these motions. One of the main results is that the crossover observed in the dynamics of the methyl groups in the HP36 sample at 170 K is absent in FMOC-leu.

Freezing of dynamics of a methyl group in a protein hydrophobic core at cryogenic temperatures by deuteron NMR spectroscopy.

Methyl groups are thought to dominate the dynamics of proteins after slow collective modes of motion freeze out in a glass-transition process. In this work we investigate methyl group dynamics of a key hydrophobic core leucine residue in chicken villin headpiece subdomain protein at 140-4 K using deuteron NMR longitudinal relaxation measurements. A distinct increase in the apparent activation energy is observed at approximately 95 K, indicating an abrupt freezing of methyl group dynamics.

Probing the dynamics of a protein hydrophobic core by deuteron solid-state nuclear magnetic resonance spectroscopy.

With the goal of investigating dynamical features of hydrophobic cores of proteins over a wide range of temperatures, the chicken villin headpiece subdomain protein (HP36) was labeled at a "single" site corresponding to any one of the two C(delta)D(3) groups of leucine-69, which is located in a key position of the core. The main techniques employed are deuteron NMR quadrupolar echo line shape analysis, and T(1Z) (Zeeman) and T(1Q) (quadrupolar order) relaxation experiments performed at 11.7 and 17.

NMR analysis of methyl groups at 100-500 kDa: model systems and Arp2/3 complex.

Large macromolecular machines are among the most important and challenging targets for structural and mechanistic analyses. Consequently, there is great interest in development of NMR methods for the study of multicomponent systems in the 50-500 kDa range. Biochemical methods also must be developed in concert to produce such systems in selectively labeled form.