An Efficient Labelling Approach to Harness Backbone and Side‐Chain Protons in <sup>1</sup>H‐Detected Solid‐State NMR Spectroscopy

Mance, Deni; Sinnige, Tessa; Kaplan, Mohammed; Narasimhan, Siddarth; Daniëls, Mark A.; Houben, Klaartje; Baldus, Marc; Weingarth, Markus

doi:10.1002/ange.201509170

Cited by 31 publications

(65 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, dramatic improvements in sensitivity have been obtained by the implementation of 1 H-detected magic angle spinning (MAS) solid-state NMR methods (46,47), especially when fast MAS is combined with perdeuteration of the samples (46,(48)(49)(50)(51). This approach is being applied to an increasing number of membrane proteins (52)(53)(54)(55).…”

Section: Introductionmentioning

confidence: 99%

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

Park

Berkamp

Radoicic

et al. 2017

Biophysical Journal

View full text Add to dashboard Cite

The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G proteincoupled receptor, located in the cell membrane of neutrophils. Here, we describe 1 H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn 2þ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

show abstract

Section: Introductionmentioning

confidence: 99%

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

Park

Berkamp

Radoicic

et al. 2017

Biophysical Journal

View full text Add to dashboard Cite

show abstract

“…One approach to access side-chain proton resonances is the fractional labeling of side chains with 2 H and 1 H, which has the advantage of protonation at many sites, but with a dramatic reduction in sensitivity and potential loss of resolution due to the presence of multiple isotopomers (37)(38)(39). These problems have limited the application of these strategies for protein structure determination.…”

mentioning

confidence: 99%

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

Andreas

Jaudzems

Staněk

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

230

288

View full text Add to dashboard Cite

Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of 1 H-1 H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.NMR spectroscopy | magic-angle spinning | protein structures | proton detection | viral nucleocapsids D espite tremendous progress in the analysis of biomolecular samples over the last two decades (1-7), routine application of magic-angle spinning (MAS) NMR in biology is still limited by the inherently low sensitivity. The direct detection of proton resonances is a straightforward way to counter this problem, but entails a trade-off with resolution due to the strong homonuclear dipolar interactions among proton nuclei. High-resolution proton-detected methods were first demonstrated with modest spinning frequencies by today's standards (∼10 kHz) and relied on a reduction of 1 H-1 H couplings by high levels of dilution with deuterium, typically perdeuteration, and complete (8, 9) or partial (10-12) protonation at exchangeable sites. The need for narrow proton resonances without such extreme levels of deuteration has motivated a continuous technological development, resulting in a dramatic increase in the available spinning frequency (13)(14)(15)(16)(17)(18)(19)(20).At MAS frequencies of 40-60 kHz, deuteration and 100% reprotonation at exchangeable sites, primarily amide protons, result in resolved and sensitive spectra, similar in quality to the case of higher dilution levels and lower spinning frequencies (21-23). This opens the way to rapid sequential assignment of backbone resonances (24-27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28-32). 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. However, a high deuteration level severely limits observation of side-chain s...

show abstract

“…In comparison to the amide backbone, the proton density in the aliphatic sidechain is significantly higher. Fractional labelling of protons in the amino acid sidechains Reif 2012, 2013;Asami et al 2010) and/or inverse fractional deuteration (Mance et al 2015) yield excellent spectral resolution at moderate MAS frequencies.…”

Section: Introductionmentioning

confidence: 99%

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR

et al. 2019

View full text Add to dashboard Cite

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, protondetection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH 3 , CH 2 D, CHD 2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.

show abstract

An Efficient Labelling Approach to Harness Backbone and Side‐Chain Protons in ¹H‐Detected Solid‐State NMR Spectroscopy

Cited by 31 publications

References 40 publications

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

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

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR

Contact Info

Product

Resources

About

An Efficient Labelling Approach to Harness Backbone and Side‐Chain Protons in 1H‐Detected Solid‐State NMR Spectroscopy

Cited by 31 publications

References 40 publications

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

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

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR

Contact Info

Product

Resources

About

An Efficient Labelling Approach to Harness Backbone and Side‐Chain Protons in ¹H‐Detected Solid‐State NMR Spectroscopy