Vipin Agarwal

Protein Side-Chain Dynamics As Observed by Solution- and Solid-State NMR Spectroscopy: A Similarity Revealed

In this paper, we seek to compare the internal dynamics of a small globular protein, SH3 domain from alpha-spectrin, in solution and in a crystalline state. The comparison involves side-chain methyl 13C R1 relaxation rates that are highly sensitive to local dynamics in the vicinity of the methyl site. To conduct the relaxation measurements, protein samples have been prepared using specially labeled alpha-ketoisovalerate precursors, resulting in selective incorporation of the 1H-13C spin pair in one or both methyl groups of the valine and leucine side chains. The sparse labeling pattern in an otherwise deuterated sample makes it possible to record high-resolution 13C, 1H solid-state spectra using magic angle spinning experiment with a MAS frequency of 22 kHz. Furthermore, this labeling scheme avoids proton-driven 13C-13C spin-diffusion effects, thus allowing for accurate measurements of 13C R1 relaxation in the individual methyl groups. While the relaxation response from a polycrystalline sample is generally expected to be multiexponential, we demonstrate both theoretically and experimentally that in this particular case the relaxation profiles are, in excellent approximation, monoexponential. In fact, solid-state relaxation data can be interpreted in a model-free fashion, similar to solution data. Direct comparison between the experimentally measured solid and solution rates reveals a strong correlation, r = 0.94. Furthermore, when solution rates are corrected for the effect of the overall molecular tumbling (quantified on the basis of the solution 15N relaxation data), the results are in one-to-one agreement with the solid-state rates. This finding indicates that methyl dynamics in the solution and solid samples are quantitatively similar. More broadly, it suggests that the entire dynamic network, including motions of side chains in the protein hydrophobic core and backbone motions, is similar. This result opens interesting possibilities for combined interpretation of solid- and solution-state relaxation data, potentially leading to a detailed characterization of internal protein dynamics on a wide range of time scales.

Residual methyl protonation in perdeuterated proteins for multi-dimensional correlation experiments in MAS solid-state NMR spectroscopy

NMR studies involving perdeuterated proteins focus in general on exchangeable amide protons. However, non-exchangeable sites contain as well a small amount of protons as the employed precursors for protein biosynthesis are not completely proton depleted. The degree of methyl group protonation is in the order of 9% for CD2H using >97% deuterium enriched glucose. We show in this manuscript that this small amount of residual protonation is sufficient to perform 2D and 3D MAS solid-state NMR experiments. In particular, we suggest a HCCH-TOBSY type experiment which we successfully employ to assign the methyl resonances in aliphatic side chains in a perdeuterated sample of the SH3 domain of chicken alpha-spectrin.

High-Resolution Double-Quantum Deuterium Magic Angle Spinning Solid-State NMR Spectroscopy of Perdeuterated Proteins

We show in this manuscript that (2)H,(13)C correlation spectra in uniformly (2)H,(13)C isotopically enriched peptides and proteins can be recorded in MAS solid-state NMR with site specific resolution. A resolved deuterium dimension is obtained by evolving (2)H double-quantum coherences. Experimental (2)H line widths are obtained that are as small as 16 Hz (0.17 ppm at 600 MHz) in the double-quantum dimension. The unprecedented resolution in the deuterium dimension obtained for proteins opens new perspectives for correlation experiments and, in particular, for the characterization of dynamics of proteins in the solid-state.

Identification of Hydroxyl Protons, Determination of Their Exchange Dynamics, and Characterization of Hydrogen Bonding in a Microcrystallin Protein

Heteronuclear correlation experiments employing perdeuterated proteins enable the observation of all hydroxyl protons in a microcrystalline protein by MAS solid-state NMR. Dipolar-based sequences allow magnetization transfers that are >50 times faster compared to scalar-coupling-based sequences, which significantly facilitates their assignment. Hydroxyl exchange rates were measured using EXSY-type experiments. We find a biexponential decay behavior for those hydroxyl groups that are involved in side chain-side chain C-O-H...O horizontal lineC hydrogen bonds. The quantification of the distances between the hydroxyl proton and the carbon atoms in the hydrogen-bonding donor as well as acceptor group is achieved via a REDOR experiment. In combination with X-ray data and isotropic proton chemical shifts, availability of (1)H,(13)C distance information can aid in the quantitative description of the geometry of these hydrogen bonds. Similarly, correlations between backbone amide proton and carbonyl atoms are observed, which will be useful in the analysis of the registry of beta-strand arrangement in amyloid fibrils.

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

The amyloid β-peptide (Aβ) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimers disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Aβ peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.

Selective 1H–1H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR

Very fast magic-angle spinning (MAS > 80 kHz) NMR combined with high-field magnets has enabled the acquisition of proton-detected spectra in fully protonated solid samples with sufficient resolution and sensitivity. One of the primary challenges in structure determination of protein is observing long-range 1H-1H contacts. Here we use band-selective spin-lock pulses to obtain selective 1H-1H contacts (e.g., HN-HN) on the order of 5-6 Å in fully protonated proteins at 111 kHz MAS. This approach is a major advancement in structural characterization of proteins given that magnetization can be selectively transferred between protons that are 5-6 Å apart despite the presence of other protons at shorter distance. The observed contacts are similar to those previously observed only in perdeuterated proteins with selective protonation. Simulations and experiments show the proposed method has performance that is superior to that of the currently used methods. The method is demonstrated on GB1 and a β-barrel membrane protein, AlkL.

Sensitivity enhancement of double quantum nmr spectroscopy by modified cpmg

A modified Carr-Purcell-Meiboom-Gill (CPMG) sequence for sensitivity enhancement of dipolar coupled homonuclear spin pairs in static solid-state NMR is presented. The modified CPMG block uses the Hahn-solid-Hahn echo as basic element of the CPMG echo train to refocus the homonuclear dipolar coupling and chemical shift anisotropy. The new CPMG sequence is dubbed as Hahn-solid-Hahn Carr-Purcell-Meiboom-Gill (HSHCPMG). We demonstrate a gain in signal to noise ratio of approximately 4.2 using HSHCPMG sequence in double quantum filtered CP experiment for 5%-(13)C(2)-(15)N-glycine. The resulting gain in sensitivity in the spikelet spectrum does not compromise the anisotropic information that is available from static NMR lineshapes. As an example, relative orientation angles of chemical shift anisotropy tensors for the alpha and carbonyl carbons in glycine are determined from the 2D DOQSY experiment recorded with the HSHCPMG block in the acquisition dimension. The resultant relative orientation angles of the two CSA tensors are compared to those obtained from 2D DOQSY experiment acquired without sensitivity enhancement as well as to the data as available from single crystal NMR experiments.