Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR

Abstract

In DNP MAS NMR experiments at ~80–110 K, the structurally important −13CH3 and −15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR line shapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3, and (5) the protonated and deuterated dipeptide N-Ac-VL over 78–300 K. In APG, the three-site hopping of the Ala-Cβ peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13C’s and 15N’s. Similarly, the 15N signal from Ala-NH3+ disappears at ~ 173 K, concurrent with the attenuation in CP experiments of other 15N’s as well as 13C’s. In bR and PI3-SH3, the methyl groups are attenuated at ~95 K, while all other 13C’s remain unaffected. However, both systems exhibit substantial losses of intensity at ~243 K. Finally, with spectra of Ala and N-Ac-VL, we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with recovering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.

Insights into protein misfolding and aggregation enabled by solid-state NMR spectroscopy

“…One interesting application is to study smaller amounts of labeled protein aggregates under dilute conditions, for instance in cellular extracts [132]. Current DNP methods are typically reliant on cryogenic temperatures, which enhance the performance of dipolar-recoupling experiments, but also limit the ability to measure highly valuable dynamics data and often result in reductions in spectral resolution [82, 133]. A complementary new development is the advent of faster MAS using ever-smaller rotor sizes, with state-of-the-art probes enabling MAS rates in excess of 100 kHz [134].…”

In Situ Characterization of Pharmaceutical Formulations by Dynamic Nuclear Polarization Enhanced MAS NMR

“…15,16 The recent developments of high-power microwave sources, NMR probes for cryogenic MAS, biradical polarizing agents, and new sample preparation methods, have enabled DNP to be successfully applied to investigate the dynamics and structures of micro-crystalline peptides, membrane proteins, amyloid fibrils, natural products, and catalytic materials. 17–29 …”

Electron Decoupling with Chirped Microwave Pulses for Rapid Signal Acquisition and Electron Saturation Recovery

“…With a5ms DNP period, electron decoupling results in a195 %increase in signal intensity.MAS at 4.3 K, DNP,electron decoupling,and short recycle delays improve the sensitivity of 13 Cinthe vicinity of the polarizing agent. [20][21][22][23][24][25] DNP transfer mechanisms require polarizing agents exhibiting electron paramagnetic resonance (EPR). [11][12][13][14] Mechanical rotation of the sample about the magic angle of 54.78 8 with respect to the magnetic field partially averages anisotropic interactions of the magnetic resonance Hamiltonian, improving spectral resolution and sensitivity.…”

“…[15][16][17][18] Often, sensitivity is also increased through the transfer of polarization from spins with stronger Zeeman interactions by radiofrequency [16,19] or microwave irradiation. [20][21][22][23][24][25] DNP transfer mechanisms require polarizing agents exhibiting electron paramagnetic resonance (EPR). [20][21][22][23][24][25] DNP transfer mechanisms require polarizing agents exhibiting electron paramagnetic resonance (EPR).…”

“…In the case of dynamic nuclear polarization (DNP), hyperfine interactions are required to transfer spin polarization from electron to nuclear spins. [20][21][22][23][24][25] DNP transfer mechanisms require polarizing agents exhibiting electron paramagnetic resonance (EPR). [26] Here, we use as table organic monoradical, trityl (Finland radical), which has ar elatively narrow EPR lineshape.…”