Simultaneous recording of intra- and inter-residue linking experiments for backbone assignments in proteins at MAS frequencies higher than 60 kHz

Sharma, Kshama; Madhu, Perunthiruthy K.; Agarwal, Vipin; Mote, Kaustubh R.

doi:10.1007/s10858-019-00292-y

Cited by 24 publications

(45 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…intraresidue correlations) which is, fortunately, often straightforward, using the hCANH and hNCAH spectra as demonstrated in Figure 2d. The spectra in Figure 2d were acquired simultaneously to improve sensitivity and maximize consistency of chemical shifts [25], [26]. The succeeding 15 N(i+1) chemical shift can then be found in the hNcoCAH spectrum and adjacent residues are simply linked over the 13 Ca resonance.…”

Section: Methodsmentioning

confidence: 99%

Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR

et al. 2020

View full text Add to dashboard Cite

Most commonly small outer membrane proteins, possessing between 8 and 12 β-strands, are not involved in transport but fulfill diverse functions such as cell adhesion or binding of ligands. An intriguing exception are the 8-stranded β-barrel proteins of the OmpW family, which are implicated in the transport of small molecules. A representative example is AlkL from Pseudomonas putida GPoI, which functions as a passive importer of hydrophobic molecules. This role is of high interest with respect to both fundamental biological understanding and industrial applications in biocatalysis, since this protein is frequently utilized in biotransformation of alkanes. While the transport function of AlkL is generally accepted, a controversy in the transport mechanism still exists. In order to address this, we are pursuing a structural study of recombinantly produced AlkL reconstituted in lipid bilayers using solid-state NMR spectroscopy. In this manuscript we present 1 H, 13 C and 15 N chemical shift assignments obtained via a suite of 3D experiments employing high magnetic fields (1 GHz and 800 MHz) and the latest magic-angle spinning (MAS) approaches at fast (60-111) kHz rates. We additionally analyze the secondary structure prediction in comparison with those of published structures of homologous proteins. Abbreviations IMAC-immobilized metal affinity chromatography, EDTA-ethylenediaminetetraacetic acid, LDAO-lauryldimethylamine N-oxide, OG-n-octyl-β-D-glucopyranoside, DMPC-1,2dimyristoyl-sn-glycero-3-phosphocholine, CP-cross polarization, INEPT-insensitive nuclei enhanced by polarization transfer, BASS-SD-band-selective spectral spin diffusion, TOCSYtotal correlation spectroscopy, MAS-magic-angle spinning

show abstract

Section: Methodsmentioning

confidence: 99%

Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Previous multi-acquisition ssNMR experiments utilized two to four pathways originating from SPECIFIC-CP or other NC transfer variants. [19][20][21][22][23][24][25][26][27][28][29][30][31][32] In contrast, the broadband SPECIFIC-CP sequence creates seven different pathways (Figure S8b). The intensities of the CANtr, CONtr, NCAtr, and NCOtr pathways, were 103 %, 72 %, 55 %, and 100 %, respectively, relative to CA and CO SPECIFIC-CP transfers (Figures S5-S6).…”

Section: Chemphyschemmentioning

confidence: 99%

“…[7][8][9][10][11][12][13][14][15][16][17][18] Additionally, the introduction of multi-acquisition polarization optimized experiments (POE) has further boosted data acquisition [19][20][21][22][23] for both solution-and ssNMR spectroscopy. [19][20][21][22][23][24][25][26][27][28][29][30][31][32] Indeed, 1 H detection has significantly improved the sensitivity of fast and ultrafast MAS experiments for fully protonated protein samples; however, the broad line widths of the 1 H resonances reduce the spectral resolution. [8,14,15,33,34] The latter is often exacerbated by conformational heterogeneity, restricting the range of applications of ssNMR spectroscopy.…”

Section: Introductionmentioning

confidence: 99%

PHRONESIS: A One‐Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid‐State NMR Spectroscopy

et al. 2022

View full text Add to dashboard Cite

Solid‐state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane‐bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, 1H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance‐specific assignments remain a bottleneck for this technique. Here, we present a comprehensive multi‐acquisition experiment (PHRONESIS) that simultaneously generates up to ten 3D 1H‐detected ssNMR spectra. PHRONESIS utilizes broadband transfer and selective pulses to drive multiple independent polarization pathways. High selectivity excitation and de‐excitation of specific resonances were achieved by high‐fidelity selective pulses that were designed using a combination of an evolutionary algorithm and artificial intelligence. We demonstrated the power of this approach with microcrystalline U‐13C,15N GB1 protein, reaching 100 % of the resonance assignments using one data set of ten 3D experiments. The strategy outlined in this work opens up new avenues for implementing novel 1H‐detected multi‐acquisition ssNMR experiments to speed up and expand the application to larger biomolecular systems.

show abstract

“…In particular, the progressive extension of the NMR spectra to the 1 H dimension by faster MAS rates has revolutionized the field, boosting both resolution and sensitivity, and thus enabling the investigation of more complex protein systems of larger molecular weight, with significantly reduced sample quantities (down to less than 0.5 mg) [2][3][4]. New efficient strategies including time-shared experiments [5][6][7], non-uniform sampling [8], projection spectroscopy [9] and paramagnetic doping [10,11] allow today the accelerated acquisitions of multidimensional spectra for resonance assignment, their rapid automatic evaluation, and their use for the measurement of site-specific dynamics parameters. Most importantly, faster MAS, often in combination with suitable deuteration schemes, reduces or suppresses the unwanted contributions of the 1 H network, permitting a quantitative analysis of spin relaxation in terms of conformational dynamics [12].…”

Section: Introductionmentioning

confidence: 99%

Protein structural dynamics by Magic-Angle Spinning NMR

Bonaccorsi

Marchand

Pintacuda

2021

Current Opinion in Structural Biology

View full text Add to dashboard Cite

Simultaneous recording of intra- and inter-residue linking experiments for backbone assignments in proteins at MAS frequencies higher than 60 kHz

Cited by 24 publications

References 52 publications

Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR

Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR

PHRONESIS: A One‐Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid‐State NMR Spectroscopy

Protein structural dynamics by Magic-Angle Spinning NMR

Contact Info

Product

Resources

About