Journal of Biomolecular NMR最新文献

Peak information extraction is an important step for all types of protein NMR experiments, from structure determination to titration experiments. In most cases, extraction of peak information, or peak-picking, is done semi-automatically or manually, relying on the spectroscopist’s expertise to guard against errors in the algorithm. Optimus Peak is designed for fully automatic peak-picking in multi-dimensional protein NMR spectra for both solid-state and solution NMR. The program shows a low false positive rate, due to its more robust evolutionary algorithm-based fitting. The program is also designed to fit together with other programs by being implemented as a terminal based command line program. Optimus Peak was tested using both solid-state and solution NMR experiments, for both 2D and 3D experiments, using 67 spectra from 12 different proteins, consisting of 26 different types of NMR experiments.

Isotopic enrichment of pharmacologically relevant protein targets is crucial for structural studies by nuclear magnetic resonance (NMR) and plays a key role in advancing structure-guided drug discovery. Many clinically important drug targets require expression in eukaryotic systems—such as mammalian, yeast, or insect cells—rather than prokaryotic hosts. This requirement limits the feasibility of high-throughput isotopic labeling and poses challenges for obtaining uniformly isotope-labeled proteins suitable for NMR analysis. While several enrichment strategies have been developed, no broadly applicable enrichment platform has emerged for eukaryotic expression systems. In this study, we introduce Cupriavidus necator as an alternative biological source for ¹⁵N and ¹³C isotopic enrichment to support protein production in eukaryotic systems. To evaluate this approach, we selected the kinase domain of EPHA2, a receptor tyrosine kinase implicated in colorectal cancer progression and an important target for therapeutic inhibitor development. Isotopic incorporation was quantified using liquid chromatography-mass spectrometry (LC-MS), revealing enrichment levels of 79% for ¹⁵N and 69% for ¹³C. These results demonstrate that Cupriavidus necator can serve as a robust and flexible platform for generating isotopically enriched biomolecules compatible with eukaryotic protein expression, thereby enabling NMR investigations of disease-relevant protein targets.

Band-selective excitation short-transient (BEST) sequences are widely used for protein NMR experiments that start with amide proton magnetization, such as ¹H-¹⁵N HSQC, ¹H-¹⁵N TROSY, and multidimensional backbone assignment experiments, because the optimization of amide proton longitudinal relaxation afforded by the BEST methodology allows for much greater sensitivity when using short scan times. Here we show that the BEST methodology can be easily incorporated in sequences for measuring proton transverse relaxation rates (¹H R₂), which are typically used to determine paramagnetic relaxation enhancements (PREs). The resulting BEST-HSQC-PRE and BEST-TROSY-PRE experiments afford similar or better sensitivity for measuring PREs compared to previous methods, provide equally accurate measurements of transverse relaxation rates (and therefore PREs), and allow shorter scan times to be used.

We demonstrate here a pulse sequence based on rotational-echo double resonance (REDOR) that can help distinguish resonances based on the (psi)-torsion angle, which allows it to distinguish (alpha)-helical and (beta)-sheet regions in solid proteins under magic-angle-spinning. The method relies on conformation-dependent differences in distances between an amide (^{1})H and amide (^{15})N nuclei not covalently attached to it. Dephasing from this remote (^{15})N nucleus is obtained in presence of the much stronger one-bond dipole-dipole coupling by using the (mathrm {theta })-REDOR sequence. Experiments are demonstrated on perdeuterated (and 70% backexchanged) sample of uniformly (^{13})C, (^{15})N labeled model protein GB1 at the magic-angle spinning frequency of 41.67 kHz. This method will be useful in simplifying chemical-shift assignments in proteins where the structure is already known, and we anticipate a direct application in determining secondary structures without relying on (^{13})C chemical-shifts.

Monoclonal antibodies (mAbs) are leading therapeutic agents due to their high specificity and limited side effects. Ensuring their structural integrity under stress and maintaining batch consistency require robust quality control. Methyl 2D NMR has emerged as a powerful tool to probe mAb structure at natural isotopic abundance, enabling spectral fingerprint comparisons across production batches to detect subtle structural changes. However, extracting atomic-level structural information requires assignment of methyl resonances to their amino acids. While such assignments are available for several antigen-binding fragments (Fabs), no comprehensive assignment has been reported for the crystallisable fragment (Fc). In this study, we present the methyl group assignment of the 50-kDa Fc fragment of an immunoglobulin G1 (IgG1) antibody. Using cell-free expression, strategic isotopic labelling, and high-quality 2D and 3D NMR experiments, we successfully assigned 94% of methyl resonances of a non-glycosylated Fc. Given that therapeutic mAbs are typically produced in Chinese Hamster Ovary (CHO) cells, we transferred this assignment to the methyl spectrum of a glycosylated Fc fragment obtained by the enzymatic cleavage of a CHO-produced mAb at natural abundance, achieving 83% assignment coverage. This assignment was then used to investigate the impact of methionine oxidation on Fc structure at atomic resolution using NMR. The methyl group assignment transforms 2D methyl NMR fingerprinting into a powerful tool for quality control. It enables the direct comparison of spectra acquired on mAbs produced at natural abundance, allowing the detection and localisation of chemical modifications and structural changes without the need for isotopic labelling. This approach offers a robust solution for monitoring the structural integrity of therapeutic antibodies throughout development and manufacturing.

The R3H domain of the human protein Sµbp-2 was produced with 5-fluoro-L-isoleucine (FIle) and 5,5-difluoro-L-isoleucine (diFIle) as probes for detection by ¹⁹F-NMR spectroscopy. The fluorinated protein, produced by cell-free protein synthesis, was obtained more easily with diFIle than FIle as FIle readily hydrolysed at pH 7.5 with the release of fluoride. The ¹⁹F-NMR spectra showed large chemical shift ranges but were heterogeneous. The heterogeneities arose from difficulties to fully exclude canonical isoleucine, the presence of multiple conformations and limited stability of the proteins, with the sample made with diFIle being particularly prone to precipitation. ¹⁹F resonance assignments were obtained by comparison of the chemical shifts of γ₁-protons with those observed in the wild-type protein. Non-uniform cross-peak intensities observed in short-delay ¹H,¹⁹F correlation experiments suggest incomplete averaging of ³J_HF couplings and therefore preferential rotamer populations of the CH₂F and CHF₂ groups.

In this communication we describe a new scheme to process the data from stimulated echo protein diffusion experiments. For a series of gradient-encoded proton spectra (:{f}_{k}left(omega:right)) considered over the selected spectral region (:({omega:}_{left},{omega:}_{right})), we build a model to approximate the unique (protein-dependent) shape of the spectrum. Taking a cue from the optimal filtration theory, (:{f}_{model}left(omega:right)) is constructed as the intensity-weighted combination of (:{f}_{k}left(omega:right)). The so obtained (:{f}_{model}left(omega:right)) is then used to fit the individual spectra (:{f}_{k}left(omega:right)), thus providing highly accurate estimates for the integral signal intensities that are subsequently used for Stejskal-Tanner-type analyses. This algorithm has been implemented as a part of a new web server, named DDfit (https://ddfit.org, mirror at https://ddfit.bio-nmr.spbu.ru/). The server accepts spectrometer data from the standard stimulated and double-stimulated echo experiments by Bruker, as well as custom-designed experiments. The server is easy to use, with data processing taking no more than several seconds. Our tests using simulated as well as experimental data found that DDfit determines protein diffusion coefficients with both accuracy and precision, offering several-fold improvement in precision compared to other processing schemes.

Owing to their high specificity and therapeutic effectiveness, monoclonal antibodies (mAbs) have rapidly become one of the leading classes of biologic drugs used to treat critical illnesses. The antigen-binding fragment (Fab) of mAbs plays a key role in the antigen recognition, so its structural characterization is essential, as even a slight change to its Higher Order Structure (HOS) can impact the antibody’s potency. Recently, 2D methyl NMR has been introduced as a powerful method to assess both the structure and integrity of therapeutic Fab fragments. However, the identification of methyl group resonances in NMR spectra remains rare since Fabs are large heterodimers of ~ 50 kDa. Here, we present the methyl group assignment of an IgG1 Fab produced in a cell-free system with an optimal isotope labelling. We first assigned 99% of the alanine, isoleucine, leucine, methionine, and valine methyl groups of the therapeutic Fab targeting LAMP1 antigen. Building on this assignment, we propose a “divide and conquer” strategy that exploits sequence identities to rapidly assign methyl groups of other IgG1 Fabs. We demonstrate that the assignment of the Fab’s constant region can easily be transferred from one IgG1 to another and that the variable part of a new Fab can be assigned using smaller uniformly ¹⁵N,¹³C-labelled constructs. We applied our strategy to ipilimumab’s Fab and, using the assignment of ipilimumab’s variable part and Fab anti-LAMP1’s constant part, we could transfer the assignment of 89% of the methyl-containing amino acids to the entire ipilimumab Fab without having to produce deuterated samples. This assignment strategy can be generalised to any other IgG1 Fabs provided that their constant regions are identical and the strategy can be adapted to accommodate the expression levels of the different variable domains. This new method drastically facilitates the Fab assignment process, making it suitable for the pharmaceutical timeline.

The efficient computer optimization of magnetic resonance pulses and pulse sequences involves the calculation of a problem-adapted cost function as well as its gradients with respect to all controls applied. The gradients generally can be calculated as a finite difference approximation, as a GRAPE approximation, or as an exact function, e.g. by the use of the augmented matrix exponentiation, where the exact gradient should lead to best optimization convergence. The calculation of the exact gradient, on the other hand, is computationally demanding and is one of the determining factors for the overall time needed for optimization. Its improved calculation, e.g. by faster analytical solutions, is therefore highly desired. The majority of pulse optimizations involve a single spin 1/2, for which propagation is either represented by 3D-rotations or quaternions. Here, highly efficient analytical solutions for gradients for both cases are derived with respect to various possible controls. Controls are either x and y pulses, but also z-controls, as well as gradients with respect to amplitude and phase of a pulse shape. In addition, analytical solutions with respect to pseudo controls, involving holonomic constraints to maximum rf-amplitudes, maximum rf-power, or maximum rf-energy, are introduced. Using the hyperbolic tangent function, maximum values are imposed in a fully continuous and differentiable way. The obtained analytical gradients allow the calculation two orders of magnitude faster than the augmented matrix exponential approach, automatically speeding up any optimization in any optimization program by the same factor for the corresponding set of controls. The use of exact gradients for different controls is finally demonstrated in a number of optimizations involving broadband pulses of potential use in biomolecular applications for (^{15})N, (^{13})C, and (^{19})F.

M9 minimal media and its enhanced variants (M9 + and M9++) are widely used for recombinant protein expression in Escherichia coli, particularly for isotopic labeling required in structural biology techniques such as NMR spectroscopy. This study investigates how different compositions of M9-based media (M9, M9+, and M9++) influence bacterial growth, metabolic stress, and central carbon metabolism during recombinant expression of the protein. Using 1D ¹H NMR spectroscopy and multivariate statistical analysis, we observed distinct media-dependent metabolic shifts. Standard M9 exhibited limited bacterial growth and heightened stress-related fermentation, indicated by high ethanol and acetate levels. In contrast, M9 + significantly increased biomass but promoted pronounced overflow metabolism. M9 + + presented intermediate biomass levels and markedly reduced overflow metabolites, favoring biosynthesis pathways, notably increasing valine, acetoin, and formate concentrations. These findings suggest that further optimization of glucose concentration, nitrogen sources, and phosphate buffering could significantly improve the metabolic balance of M9++, creating an enhanced medium tailored for efficient, high-quality recombinant protein expression and isotopic labeling in E. coli.