Journal of Biomolecular NMR最新文献

We present a novel approach for side-chain-selective deuteration of proteins to improve ¹H_α spectral resolution and to simplify side-chain signals in ¹H-detected protein solid-state NMR (SSNMR) with a simple bio-expression method using E. coli BL21 (DE3). ¹H-detected SSNMR using ultra-fast magic-angle spinning (MAS) at a spinning rate of 60 kHz or higher is attracting attention as a powerful method of protein structure determination. However, even with ultra-fast MAS at 100 kHz, the ¹H line broadening due to ¹H-¹H dipolar interactions cannot be eliminated, posing an obstacle to signal assignment and structure determination. To improve resolution for SSNMR-based protein structural analysis, we developed a method to selectively deuterate side-chains at a high deuteration level while maintaining the protons at the α-position. This selective labeling method is based on the transamination reaction in the amino-acid biosynthesis pathway and switching a medium from an unlabeled H₂O medium containing D-glucose (glucose), ammonium chloride, and amino acid mixture for rapid cell growth to a labeled H₂O medium containing [²H, ¹³C]-glucose, ¹⁵N-labeled ammonium chloride, and a [²H, ¹³C, ¹⁵N]-labeled amino-acid mixture just before the induction. With [²H, ¹³C]-labeled glucose and a [²H, ¹³C, ¹⁵N]-labeled amino-acid mixture as the carbon sources, this medium-switching method provides a simple and efficient means to express a selectively deuterated protein GB1 domain (GB1) sample, which is achieved by promoting efficient back-protonation at the α-position via the transamination reaction while retaining side-chain deuterons to a large extent. The yield of the GB1 protein was found to be enhanced by a factor of ca. 1.5 with the medium-switching method, compared with that for the expression with a traditional M9 minimal medium in H₂O without medium-switching. For the selectively deuterated GB1 sample, the resultant ¹H resolution for resolved ¹H_α peaks in ¹H-detected 2D ¹H/¹³C correlation SSNMR at a MAS rate of 70 kHz was improved by a factor of 1.21 on average, compared with the corresponding resolution for a fully protonated, uniformly ¹³C- and ¹⁵N-labeled GB1 sample. Furthermore, side-chain signal assignment is facilitated by utilizing residual protons of the side chains. Our results also suggest that the side-chain deuteration level can be altered by adjusting the level of the deuterated amino-acid mixture in the expression system.

The use of ¹H detection, made possible by very fast magic-angle spinning (MAS), has revolutionized the field of biomolecular solid-state NMR. In the past, ¹H detection was often paired with deuteration schemes to achieve the highest possible resolution needed for protein structural characterization. However, with modern probes capable of MAS rates over 100 kHz, deuteration is no longer required, resulting in a need to measure long-range distances in fully protonated systems. In this study, we evaluate the potential of two 3D pulse sequences, (H)NCOH and (H)NCAH, to measure long-range C-H correlations in a fully protonated protein sample at a MAS rate of 105 kHz. Our results show that the (H)NCOH spectrum contains multiple sequential and structurally relevant long-range CO–H contacts for each residue, capturing H^N contacts up to 6 Å despite transfers to side chain protons. Conversely, the (H)NCAH spectrum yields fewer Cα-H^N correlations, with those present mostly from intraresidue aliphatic proton contacts. Therefore, in protonated proteins, the extensive ¹H network leads to dipolar truncation in the Cα-H experiment, while the CO–H correlations observed are comparable to those in deuterated samples. These findings highlight the feasibility of conducting distance measurements based on long-range cross polarization, on more accessible and affordable samples, expanding the scope of proton detection for systems where deuteration and back-exchange are not possible.

Cellular functions require biomolecules to transition among various conformational sub-states in the energy landscape. A mechanistic understanding of cellular functions requires quantitative knowledge of the kinetics, thermodynamics, and structural features of the biomolecules experiencing exchange between several states. High-power Relaxation Dispersion (RD) NMR experiments have proven very effective for such measurements if the exchange occurs in timescales ranging from microseconds to milliseconds. However, scanning the significantly larger kinetic window within the time limit of instrumental availability and sample stability requires careful optimization of experiments. Understanding biomolecular functions at a mechanistic level depends on fitting such experimental data to theoretical models. However, the reliability of the fit parameters depends on the measurement schemes and is sensitive to experimental noise. Here, we benchmark different measurement schemes along with theoretical models for sub-millisecond timescale exchange and determine the robustness of these models in providing information when the measurements contain noise. Our results show that kinetics can be measured reliably from such experiments. The structural features of the exchanging sub-states, encoded in the chemical shift differences between the states, can be fitted, albeit with significant uncertainties. Information about the minor states is difficult to obtain exclusively from the RD data due to large uncertainties and sensitivity to noise.

Biomolecular dynamics in the microsecond-to-millisecond (µs–ms) timescale are linked to various biological functions, such as enzyme catalysis, allosteric regulation, and ligand recognition. In solution state NMR, Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experiments are commonly used to probe µs–ms timescale motions, providing detailed kinetic, thermodynamic, and mechanistic information at the atomic level. For investigating conformational dynamics in high-molecular-weight biomolecules, methyl groups serve as ideal probes due to their favorable relaxation properties, and ¹³C CPMG relaxation dispersion is widely employed for characterizing dynamics in selectively ¹³CH₃-labeled samples. However, conventional schemes that apply CPMG pulses with constant phase are susceptible to artifacts arising from off-resonance effects, radiofrequency (RF) field inhomogeneity and pulse imperfections. In this work we present an optimized¹³C single-quantum (SQ) CPMG experiment incorporating the [0013]-phase cycling scheme, and demonstrate its enhanced robustness against various adverse effects. Moreover, the optimized pulse scheme enables finer sampling of CPMG pulsing frequencies and is suited for studying systems with variable J_CH scalar coupling constants, thereby facilitating comprehensive characterization of µs–ms timescale dynamics of biomolecules with increased precision.

The E. coli γ-clamp loader is a 200 kDa pentameric AAA + ATPase comprised of γ, δ and δ′ subunits in a 3:1:1 ratio, which opens the ring shaped β-clamp homodimer and loads it onto DNA in a process essential for DNA replication. The clamp loading is initiated by ATP binding, which induces conformational changes in the clamp loader allowing it to bind and open the β-clamp. This is followed by DNA primer-template binding, ATP hydrolysis, and clamp release onto DNA. Despite a wealth of structural and functional data, dynamics and interactions of the γ-clamp loader and the β-clamp underlying elementary steps of this process remain elusive. Here we employed a “divide-and-conquer” strategy for the initial NMR characterization of the γ-clamp loader. A new protocol for the clamp loader assembly was proposed allowing selective incorporation of the isotope-labeled δ and δ′ subunits for NMR studies. The nearly complete ¹H, ¹⁵N and ¹³C NMR resonance assignments were obtained for the isolated modular domains of the δ and δ′ subunits, which facilitated the assignments of the full-length subunits, and side-chain methyl assignments of the subunits in the context of pentameric γ-clamp loader. NMR chemical shift analysis using the random coil index approach revealed increased flexibility in the ATP, DNA, and β-clamp binding interfaces of the isolated subunits, highlighting a potential significance of conformational dynamics for the clamp loading process. The reported clamp loader assembly protocol and resonance assignments enable the detailed NMR studies of protein dynamics and mechanochemistry of the clamp loading cycle.

Cellulose nanocrystals (CNCs) is synthesized from alpha-cellulose by acid hydrolysis method, and formation of nanocrystallization is comprised by using various microscopic and spectroscopic techniques like PXRD, XPS, Raman, FTIR, PL, UV-Vis, DSC, TGA, DLS, SEM, TEM. Nanocrystalline cellulose shows a notably higher photoluminescence (PL) intensity than cellulose, which enhances its ability to absorb and emit visible light. This increase in PL intensity is attributed to a smaller particle size of CNCs, greater surface area, and quantum confinement effects. The higher intensity of the XPS spectrum further supports the larger surface area of CNCs. PXRD and Raman spectroscopy results show that CNCs has a higher crystallinity index than cellulose. Through deconvolution of the ¹³C CP-MAS SSNMR spectrum, we confirmed a significant reduction in the relative abundance of the amorphous region of cellulose (43.61%) to just 4.97% in CNCs. The ¹³C CP-MAS SSNMR spectrum of CNCs, at the C4, C6, C2C3C5 nuclei sites, can be fitted by two distinct lines for both amorphous and crystalline region, indicating the formation of a co-crystal from two nanocrystallites. Despite this, the principal components of the CSA (chemical shift anisotropy) tensor remain unchanged, suggesting similar electronic environments for these two nanocrystallites. The spin-lattice relaxation time and local correlation time of cellulose and CNCs are determined for chemically distinct carbon nuclei residing on D-glucopyranose units. It is noteworthy that the ¹³C spin-lattice relaxation time and ¹³C local correlation time are longer for each chemically distinct nucleus in CNCs compared to cellulose. It can be predicted by observing the NMR relaxometry data that the longer relaxation time in CNCs is due to the enhancement of crystallinity index. Hence, a correlation between the crystallinity index and nuclear spin dynamics can be established by NMR relaxometry measurements. These findings offer significant insights into the intricate structure and dynamic behavior of cellulose and nanocrystalline cellulose (CNCs), crucial for advancing biomimetic material design, which has huge applications across the pharmaceutical, textile, and cosmetics industries.

Recent advances in high power NMR relaxation dispersion experiments have significantly enhanced our ability to study fast µs timescale motions in proteins, which are crucial for understanding their biological functions. Here, we have extended the detectable time window of such fast dynamics with the development of extreme power ¹H Carr-Purcell-Meiboom-Gill (¹H E-CPMG) experiments targeted at the backbone amide protons (¹H_N). Using this methodology, artifact-free relaxation dispersion profiles can be obtained up to extreme pulsing conditions with minimal setup effort using commonly used standard NMR hardware. We demonstrate the utility of ¹H E-CPMG on human ubiquitin, revealing that the previously reported peptide flip motion influences a larger region of the protein backbone than previously recognized. Additionally, we directly observed a faster dynamic process at residue T09, aligning with previously predicted pincer mode motion. These findings underscore the effectiveness of ¹H E-CPMG in extending the temporal resolution at which biologically relevant fast protein dynamics can be studied.

We show that water saturation leads to deleterious losses in sensitivity of methyl signals in selectively methyl-[¹³CH₃]-labeled protein samples of high molecular weight proteins dissolved in H₂O. These losses arise from efficient cross-relaxation between methyl protons and proximal labile protons in the protein structure. A phenomenological model for analysis of methyl intensity decay profiles that involves exchange saturation transfer of magnetization from localized proton spins of water to various labile groups in the protein structure that, in turn, efficiently cross-relax with protons of methyl groups, is described. Analysis of methyl intensity decay profiles with this model allows cross-relaxation rates (σ) between methyl and labile protons to be determined and permits identification of methyl sites in close proximity to labile groups in the protein structure.