J. Am. Chem. Soc., 2010, 132 (28), pp 9561–9563
Rapid Acquisition of Multidimensional Solid-State NMR Spectra of Proteins Facilitated by Covalently Bound Paramagnetic Tags
Philippe S. Nadaud, Jonathan J. Helmus, Ishita Sengupta and Christopher P. Jaroniec
We describe a condensed data collection approach that facilitates rapid acquisition of multidimensional magic-angle spinning solid-state nuclear magnetic resonance (SSNMR) spectra of proteins by combining rapid sample spinning, optimized low-power radio frequency pulse schemes and covalently attached paramagnetic tags to enhance protein 1H spin−lattice relaxation. Using EDTA-Cu2+-modified K28C and N8C mutants of the B1 immunoglobulin binding domain of protein G as models, we demonstrate that high resolution and sensitivity 2D and 3D SSNMR chemical shift correlation spectra can be recorded in as little as several minutes and several hours, respectively, for samples containing 0.1−0.2 μmol of 13C,15N- or 2H,13C,15N-labeled protein. This mode of data acquisition is naturally suited toward the structural SSNMR studies of paramagnetic proteins, for which the typical 1H longitudinal relaxation time constants are inherently a factor of at least 3−4 lower relative to their diamagnetic counterparts. To illustrate this, we demonstrate the rapid site-specific determination of backbone amide 15N longitudinal paramagnetic relaxation enhancements using a pseudo-3D SSNMR experiment based on 15N−13C correlation spectroscopy, and we show that such measurements yield valuable long-range 15N−Cu2+ distance restraints which report on the three-dimensional protein fold.
J. Am. Chem. Soc., 2010, 132 (29), pp 9952–9953
Validation of a Lanthanide Tag for the Analysis of Protein Dynamics by Paramagnetic NMR Spectroscopy
Mathias A. S. Hass, Peter H. J. Keizers, Anneloes Blok, Yoshitaka Hiruma and Marcellus Ubbink
Paramagnetic lanthanide tags potentially can enhance the effects of microsecond to millisecond dynamics in proteins on NMR signals and provide structural information on lowly populated states encoded in the pseudocontact shifts. We have investigated the microsecond to millisecond mobility of a two-point attached lanthanide tag, CLaNP-5, using paramagnetic 1H CPMG relaxation dispersion methods. CLaNP-5 loaded with Lu3+, Yb3+, or Tm3+ was attached to three sites on the surface of two proteins, pseudoazurin and cytochrome c. The paramagnetic center causes large relaxation dispersion effects for two attachment sites, suggesting that local dynamics of the protein at the attachment site causes mobility of the paramagnetic center. At one site the relaxation dispersions are small and limited to the immediate environment of the tag. It is concluded that paramagnetic relaxation dispersion could represent a sensitive method to probe protein dynamics. However, the selection of a rigid attachment site is of critical importance.
J. Am. Chem. Soc., 2010, 132 (29), pp 9956–9957
Solid-State 13C NMR Assignment of Carbon Resonances on Metallic and Semiconducting Single-Walled Carbon Nanotubes
Chaiwat Engtrakul*†, Mark F. Davis†, Kevin Mistry†, Brian A. Larsen†, Anne C. Dillon†, Michael J. Heben‡ and Jeffrey L. Blackburn*†
Solid-state 13C NMR spectroscopy was used to investigate the chemical shift of nanotube carbons on m- and s-SWNTs (metallic and semiconducting single-walled nanotubes) for samples with widely varying s-SWNT content, including samples highly enriched with nearly 100% m- and s-SWNTs. High-resolution 13C NMR was found to be a sensitive probe for m- and s-SWNTs in mixed SWNT samples with diameters of 1.3 nm. The two highly enriched m- and s-SWNT samples clearly exhibited features for m- and s-SNWT 13C nuclei (123 and 122 ppm, respectively) and were successfully fit with a single Gaussian, while five mixed samples required two Gaussians for a satisfactory fit.
J. Am. Chem. Soc., 2010, 132 (29), pp 9979–9981
Probing Slow Protein Dynamics by Adiabatic R1ρ and R2ρ NMR Experiments
Silvia Mangia, Nathaniel J. Traaseth, Gianluigi Veglia, Michael Garwood‡ and Shalom Michaeli
Slow μs/ms dynamics involved in protein folding, binding, catalysis, and allostery are currently detected using NMR dispersion experiments such as CPMG (Carr−Purcell−Meiboom−Gill) or spin-lock R1ρ. In these methods, protein dynamics are obtained by analyzing relaxation dispersion curves obtained from either changing the time spacing between 180° pulses or by changing the effective spin-locking field strength. In this Communication, we introduce a new method to induce a dispersion of relaxation rates. Our approach relies on altering the shape of the adiabatic full passage pulse and is conceptually different from existing approaches. By changing the nature of the adiabatic radiofrequency irradiation, we are able to obtain rotating frame R1ρ and R2ρ dispersion curves that are sensitive to slow μs/ms protein dynamics (demonstrated with ubiquitin). The strengths of this method are to (a) extend the dynamic range of the relaxation dispersion analysis, (b) avoid the need for multiple magnetic field strengths to extract dynamic parameters, (c) measure accurate relaxation rates that are independent of frequency offset, and (d) reduce the stress to NMR hardware (e.g., cryoprobes).