Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

Abstract

Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.

Proton-detected MAS NMR experiments based on dipolar transfers for backbone assignment of highly deuterated proteins

“…For the sample with 20% reprotonation level we acquired spectra on the 800 MHz spectrometer at 20 kHz MAS and on the 600 MHz spectrometer using an MAS rate of 28 kHz. [10,15,18,21,34], although a direct comparison is not possible due to significant variations of the experimental conditions. The line width dependence on the protonation level at different magnetic fields is summarized in Table 1.…”

“…Fig. 6 compares the efficiency of the above introduced HCACO-CANH, HCOCACONH and HCOCAH experiments and presented earlier pulse sequences: (i) HCOCACONH based on out-and-back CO-CA INEPT transfer [54] and (ii) HCACONH and HCOCANH experiments employing DREAM for magnetization transfer between CO and CA spins [21]. All heteronuclear transfers in these pulse schemes were achieved by CP.…”

“…Even though deuterated proteins have only been studied for a relatively short time by solid-state NMR [19,20], already a plethora of different methods for 1 H, 13 C and 15 N assignment exists [15,18,[21][22][23][24][25][26]. These approaches can be classified into two categories according to the protonation level and the accompanied typical experimental parameters: (1) fully protonated proteins [21,26,27] or perdeuterated proteins with complete reprotonation on exchangeable sites [15,18], which require ultrafast MAS frequencies (40-60 kHz) and very high external magnetic fields; (2) perdeuterated proteins with partial reprotonation on exchangeable sites (ca. 20-30%) [22][23][24][25], which can be studied even at low external magnetic fields of 9.4 T and moderately high MAS rates between 20 and 32 kHz, achievable with 3.2 mm and 2.5 mm probe heads.…”

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

“…This opens the way to rapid sequential assignment of backbone resonances (24)(25)(26)(27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28)(29)(30)(31)(32). A further increase in the MAS frequency to 100 kHz allows resonance assignment (20), a structure determination of a model protein (16), and interaction studies (15) with as little as 0.5 mg of sample.…”

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

“…14 In all structural models, Ab peptides are arranged in a parallel fashion. An antiparallel arrangement is only observed for the Iowa mutant Ab-D23N, 15 and for truncated forms of the peptide such as Ab [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] . 16 The fibril structure is stabilized in particular by hydrogen bonding interactions.…”