Exploring Protein Structures by DNP-Enhanced Methyl Solid-State NMR Spectroscopy

Mao, Jiafei; Aladin, Victoria; Jin, Xinsheng; Leeder, Alexander J.; Brown, Lynda J.; Brown, Richard C. D.; He, Xiao; Corzilius, Björn; Glaubitz, Clemens

doi:10.1021/jacs.9b11195

Cited by 29 publications

(40 citation statements)

References 97 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently the effect of spontaneous 1 H-13 C polarization transfer via heteronuclear cross-relaxation due to fast reorientation dynamics in a 13 C or 15 N MAS direct polarization experiment under DNP was discovered (Daube et al, 2016;Hoffmann et al, 2017aHoffmann et al, , 2017bPark et al, 2020). Further investigation showed that cross-relaxation-promoting methyl groups and the resulting Specific Cross Relaxation Enhancement by Active Motions under DNP (SCREAM-DNP) can be used as a tool for selective probing in biomolecules such as RNA and proteins (Mao et al, 2019). This allows to overcome the drawback of typical unspecific DNP experiments such as cross polarization (CP) and direct polarization (DP), using dynamically active methyl groups as pinpoint sources of polarization.…”

Section: Introductionmentioning

confidence: 99%

Heteronuclear Cross-Relaxation under Dynamic Nuclear Polarization in Nicotine and Caffeine

et al. 2020

Self Cite

View full text Add to dashboard Cite

Dynamic nuclear polarization (DNP) is a technique in magic-angle spinning (MAS) nuclear magnetic resonance (NMR) which leads to sensitivity enhancement and helps to overcome the issue of low polarization in detected nuclei. Recent research showed, that methyl groups, which show active reorientation dynamics and cause heteronuclear cross relaxation at typical DNP temperatures around 100 K, may be used as a pinpoint source of polarization for selective and site-specific probing. In this study, we investigated the cross-relaxation behavior of methyl groups in nicotine and caffeine under DNP. These effects could be useful for investigating receptor/ligand binding.

show abstract

Section: Introductionmentioning

confidence: 99%

Heteronuclear Cross-Relaxation under Dynamic Nuclear Polarization in Nicotine and Caffeine

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, it should be noted that the trapping efficiency of the O-state is relatively low (see Figure 43), so it would be anyway difficult to identify small changes. The unperturbed chemical shift of C18 in KR2 K/L-state is similar to what is observed in GPR K-state studied by Mao et al 176 The differences observed between KR2 dark and K/L-state (light 110 K) spectra are that the peak height ratio between C18 and C16/17 changes (Figure 62). In the dark state, the ratio of C16/17 to C18 (C16/17:C18) is nearly 1 for 1 s buildup time and less than 1 (~0.8) for 5 s and 10 s buildup time with a similar linewidth.…”

supporting

confidence: 85%

“…The CR polarization transfer pathway results in a negative enhancement, which is distinguishable from the enhanced 13 C signal, derived from conventionally used cross polarization pathway and becomes even more efficient in the presence of paramagnetic nuclei such as Gd 3+ . 207 The use of dynamics-derived CR in the DNP hyperpolarization transfer pathway, called "specific cross-relaxation enhancement by active motions under DNP" (SCREAM-DNP) 15 , has been demonstrated in several biological systems, including Interleukin-1 β 207 , RNA aptamer 15 , ribozyme 208 , and GPR 176 . Aladin et al applied active methyl groups bearing tetracycline as a ligand to probe its binding and affinity to the RNA aptamer.…”

Section: Introductionmentioning

confidence: 99%

“…15 Mao et al applied CE-DNP and 1 H- 13 C hNOE based on 13 C-methyl labeled retinal dynamics to investigate the difference in molecular packing environment surrounding the chromophore in GPR resting and K-state. 176 Together with CE-DNP and Methyl 1 H- 13 C hNOE, based on 13 C- 13 C SD, the signal of the 13 C labeled spin nearby can be selectively hyperpolarized. Therefore, the hyperpolarization pathway through fast rotating methyl groups can be used to simplify the complicated NMR spectra.…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, for GPR, only the signal from C18 was observed whereas in KR2 the signals from both C16/17 and C18 were visible at the same buildup time (1 s). 176 This suggests a more relaxed packing of the β-ionone ring in KR2 compared to GPR. From the pentameric crystal structure (6REW), KR2 has a large retinal binding pocket containing 4 water molecules.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR

Jakdetchai¹

View full text Add to dashboard Cite

KR2 is a light-driven sodium ion pump found in marine flavobacterium Krokinobacter Eikastus. The protein belongs to the microbial rhodopsin family, which is characterized by seven transmembrane helices and a retinal cofactor covalently bound to a conserved lysine residue through a Schiff base linkage. Specific features of KR2 and other sodium pumping rhodopsins are the NDQ motif, the N-terminal helix capping the protein at the extracellular side, and the sodium ion bound at the protomer interface in the pentameric structure. The ability to pump sodium ions was a surprising discovery since the positive charge at the Schiff base was long thought to hinder the transport of non-proton cations and the Grotthuss mechanism could not be applied to explain the Na+ transport. The photocycle of KR2 revealed by flashed photolysis and ultrafast femtosecond absorption spectroscopy consists of consecutive intermediates, named K, L, M, and O. Here, DNP-enhanced ssNMR was used to analyze various aspects of these intermediate states. The K/L-state can be generated and trapped by in-situ illumination inside the magnet at 110 K. The trapping of L-state together with the K-state at this temperature is unexpected as this usually leads to the trapping of only K-state in bacteriorhodopsin (BR), proteorhodopsin (PR), and channelrhodopsin 2 (ChR2). This observation suggests a lower energy barrier between K- and L-state in KR2. For the O-state, the intermediate was generated by illuminating outside the magnet, followed by rapid freezing in liquid nitrogen and transfer to the magnet. Based on these procedures, the retinal conformation, and the electrostatic environment at the Schiff base in KR2 dark, K-, L- and O-intermediates were probed using 13C-labeled retinals bound to 15N-labeled KR2 by both 1D and 2D magic angle spinning (MAS) NMR experiments. The obtained data show an all-trans retinal conformation with the distortion of 150° at H-C14-C15-H in the dark state whereas the retinal has a 13-cis, 15-anti conformation in the K- and L-state after light activation. Differences between K- and L-intermediates were observed. The retinal chemical shifts of the K-state show a large deviation from the model compound behavior between the middle and end part of the polyene chain. In the L-state, these differences are much less pronounced. These observations indicate that the light energy stored in the K-state dissipates into the protein in the subsequent photointermediate states. Furthermore, an additional shielding observed for C14 in L-state indicates the slight rotation toward a more compact 13-cis, 15-syn conformation. The distortion of the H-C14-C15-H angle in the L-state (136°) is larger than in the dark state. This twist of the retinal in the L-state would play an important role in lowering the pKa of the Schiff base, which is a prerequisite for the proton transfer from the Schiff base to the proton acceptor (D116). The electrostatic environments at the Schiff base in K- and L-states cause a de-shielding of the 15N nitrogen compared to the dark state. This indicates a stepwise stronger interaction with the counterion as the Schiff base proton moves away from the Schiff base and comes closer to the D116 in the transition from K- to L-state and approaches the proton transfer step during the M-state formation. In the O-state, the retinal was found to be in the all-trans conformation but differed to the dark state in the C13, C20, and Schiff base nitrogen chemical shifts. The largest effect (9 ppm) was observed for the Schiff base nitrogen, which could be explained by the effect of the positive charge of bound Na+ near the Schiff base in the O-state, coordinated by N112 and D116 as observed in the O-state crystal structure in the pentameric form. The structural change at the opsin followed the retinal isomerization and the energy transfer from the chromophore to the surrounding were also investigated in this thesis using various amino acids labeling schemes. Moreover, 1H-13C hNOE in combination with CE-DNP was applied to probe the dynamics of retinylidene methyl groups and 23Na MAS NMR was employed to detect the bound sodium ion at the protomer interface in KR2 dark state.

show abstract

Specific Signal Enhancement on an RNA‐Protein Interface by Dynamic Nuclear Polarization

Aladin

Sreemantula

Biedenbänder

et al. 2023

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

Sensitivity and specificity are both crucial for the efficient solid-state NMR structure determination of large biomolecules. We present an approach that features both advantages by site-specific enhancement of NMR spectroscopic signals from the protein-RNA binding site within a ribonucleoprotein (RNP) by dynamic nuclear polarization (DNP). This approach uses modern biochemical techniques for sparse isotope labeling and exploits the molecular dynamics of 13 C-labeled methyl groups exclusively present in the protein. These dynamics drive heteronuclear cross relaxation and thus allow specific hyperpolarization transfer across the biomolecular complex's interface. For the example of the L7Ae protein in complex with a 26mer guide RNA minimal construct from the box C/D complex in archaea, we demonstrate that a single methyl-nucleotide contact is responsible for most of the polarization transfer to the RNA, and that this specific transfer can be used to boost both NMR spectral sensitivity and specificity by DNP.

show abstract

Exploring Protein Structures by DNP-Enhanced Methyl Solid-State NMR Spectroscopy

Cited by 29 publications

References 97 publications

Heteronuclear Cross-Relaxation under Dynamic Nuclear Polarization in Nicotine and Caffeine

Heteronuclear Cross-Relaxation under Dynamic Nuclear Polarization in Nicotine and Caffeine

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR

Specific Signal Enhancement on an RNA‐Protein Interface by Dynamic Nuclear Polarization

Contact Info

Product

Resources

About