Journal of Biomolecular NMR最新文献

We introduce 2D Q-BEST NMR as a quantitative approach for determining protein concentrations and relative state populations from a set of three ¹H–¹⁵N correlation spectra. The BEST methodology ensures short overall experimental times and highest sensitivity. The method was validated using ubiquitin as a model system, where a precision of approximately 15–20% was achieved at the level of individual cross-peaks. This precision can be further improved by averaging corrected intensities of well-resolved resonances corresponding to the same protein state. The approach was subsequently applied to the quantification of protein concentrations across multiple samples and to the determination of conformational state populations in proteins exhibiting intrinsic or ligand-induced heterogeneity. In both cases, 2D Q-BEST NMR provides accurate and robust quantification of NMR-visible species, demonstrating its utility as a general spectroscopic tool for quantitative biomolecular NMR analysis.

Side chain assignments are essential for in-depth characterization of proteins. We present here two refined TOCSY experiments based on the use of a variant of “perfect echo” to partially suppress the negative effects arising from homonuclear ¹³C couplings in the indirect dimension: The iPE-(H)CCH-TOCSY for correlating aliphatic and aromatic side chains and the iPE-(H)C(CCO)NH-TOCSY for linking side chains, in particular methyl groups, to the backbone. The iPE-TOCSY pulse sequences result in spectra of higher sensitivity and higher resolution at the same time compared to widely used standard experiments. Higher sensitivity is achieved by delayed dephasing of the signal due to homonuclear couplings and via the COSY-type C-C transfer inherent in PE-schemes. For a protein like MBP (43 kDa), sensitivity increases of up to 3-fold are shown. For enhancing resolution, the iPE scheme allows prolonging the effectively usable t₁ time from 11 to 16 ms, which leads to sharper lines and reduces overlap significantly. These two experiments have the potential to increase the size limit for side chain assignments and simplify spectral analysis by reducing overlap.

Paramagnetic NMR and EPR spectroscopy are important biophysical methods in structural biology, and site-specific protein labeling is generally required for their application in biological systems. Thiol chemistry is widely used for site-specific paramagnetic labeling due to its high reactivity, selectivity, and efficiency. Given that more than 30% of proteins require metal ions as cofactors, the compatibility of current thiol-based labeling strategies with metalloproteins containing cysteine-coordinated metal centers requires careful evaluation. Here, we evaluated widely used thiol-based paramagnetic labeling strategies with zinc-binding proteins using the baculovirus IAP repeat (BIR) domain of X-linked inhibitor of apoptosis protein (XIAP) as a model system. The XIAP BIR domain coordinates a zinc ion through three cysteine residues and one histidine residue, forming a zinc finger motif. The reactivity of the zinc-coordinated cysteines toward different paramagnetic tags was evaluated by high-resolution mass spectrometry and NMR spectroscopy. Our results show that the commonly used thiol-reactive groups methanethiosulfonate and maleimide are incompatible with site-specific paramagnetic labeling of zinc-finger proteins, because they react with zinc-coordinating cysteines in a stoichiometry-dependent manner. In contrast, the pyridylsulfonyl thiol-reactive group shows no detectable reactivity toward zinc-coordinating cysteines and represents an ideal moiety for site-specific labeling. The metal-chelating moiety in a paramagnetic tag, like strong open-chain chelating ligand, tends to displace zinc ions from the metalloproteins, thereby exposing the coordinated cysteines for modification. Pre-saturation of these metal-chelating tags with zinc ion enables complete, site-specific labeling without perturbing the native zinc finger motif. Overall, we examined the performance of the commonly used thiol-based reactive groups on paramagnetic labeling of metalloproteins, and established a practical protocol for thiol-based site-specific labeling of metalloproteins, particularly zinc-finger proteins.

Under inflammatory conditions, the ubiquitin-like modifier FAT10 serves as a tag for protein degradation by the 26S proteasome. FAT10 is degraded along with its substrates and this process is independent of the segregase VCP/p97, which, in the regular ubiquitin pathway of degradation, is required if a substrate lacks a disordered initiation region. FAT10 itself is loosely folded and its tendency to aggregate has complicated investigations of its structure, interaction, and function. Recently, hydrogen-deuterium exchange in combination with mass spectrometry has suggested that, in preparation of degradation by the proteasome, the adapter protein NUB1 traps FAT10 in a mostly unfolded state by capturing a β-strand. β-strand capture was subsequently confirmed by magic-angle spinning (MAS) NMR spectroscopy of a stabilized variant of the N-domain of FAT10 in complex with NUB1L, the longer splice variant of NUB1. MAS NMR, in addition, revealed that the N-domain of FAT10 and NUB1L form a fuzzy complex and that the N-terminus of FAT10 is positioned for initiation of degradation by specific non-covalent interaction with NUB1L. Here, we report the investigation of the wild-type N-domain of FAT10 by MAS NMR. Co-sedimentation with NUB1L yields high-quality spectra, which enable sequential assignment of resonances. Based on MAS NMR data, the complexes of the wild-type and stabilized N-domain of FAT10 with NUB1L appear identical. The N-terminal residue of FAT10 again shows up prominently in the spectra, even though it is this time an Ala, not a Gly. Our experiences suggest that co-sedimentation in combination with MAS NMR is generally helpful in the exploration of conditional folds of intrinsically disordered proteins.

The rotational correlation time ((:{tau}_{c})) is a one of the fundamental parameters for characterizing biomacromolecules in solution. The [¹⁵N, ¹H]-TRACT (TROSY for Rotational Correlation Times) NMR experiment is a widely utilized method for determining (:{tau}_{c}) by measuring the differential ¹⁵N−¹H transverse relaxation rates of α and β states. However, extracting accurate relaxation rates from these experiments often requires researchers to rely on ad hoc scripts or tedious workflows across multiple software packages, which can introduce systematic errors during peak integration and baseline correction. To address this bottleneck, we introduce pyTRACTnmr, an open-source, modular Python application featuring a modern graphical user interface designed specifically for streamlined analysis of [¹⁵N, ¹H]-TRACT data. Built on the PySide6 framework and leveraging the nmrglue library for robust data ingestion, the software provides an interactive environment for visual baseline correction, precise integration window definition, and non-linear least-squares fitting of relaxation decays. By replacing rigid automated processing with a user-friendly, visually guided workflow, pyTRACTnmr minimizes noise amplification and ensures highly reproducible analysis, and significantly simplifies the workflow.

Relaxation phenomena greatly influence magnetic resonance experiments, from setting repetition rates and measurement times through to measuring internal motion of molecules. RelCalc provides a convenient and efficient means for symbolic and numerical evaluation of relaxation rates using Bloch, Wagness and Redfield (BWR) theory. Users supply interactions within a spin system arranged appropriately in the molecular frame. The effects of optional fluctuations around a symmetry axis, and then under either isotropic or anisotropic global tumbling are then computed. Relaxation rates linking pairs of Liouvillian basis operators are then returned symbolically, formatted as a sum of weighted reduced spectra density functions. Parameters are then supplied to replace symbolic quantities for values for rapid computation of numerical rates. We demonstrate the utility of the software by exploring relaxation interference (TROSY) effects and ’long-lived states’ in (X_2) and (AX_n) systems, which, for n=1,2, and 3, describe NH, NH(_2) and CH(_3) groups in proteins, and small molecules with n=4,6,8,12 and 20 arranged as platonic solids. The implementation is highly efficient with 65,536 relaxation rates required to perform complete Liouvillian simulation of (AX_3) methyl groups rotating about a symmetric axis in the presence of adjacent static ‘external’ spins being symbolically calculated in a few seconds on a single processor on a 2021 laptop. From this result, a complete set of numerical rates are then computed in under one second. RelCalc is implemented in python and is freely available.

β-sandwich domains of the intermediate immunoglobulin fold (IgI) are key building blocks of many large, multidomain proteins – such as titin. The uniformity of the basic fold of these domains does not seem to be an impediment to their adaptation to a wide variety of functions. IgI domains in different regions of titin have to fulfil distinct functions which is seen in subtle differences of sequence, structure and stability. This is particularly true for the N2A region whose IgI domains are subtly distinct from other parts of I-band titin. We have already shown the unusual structure and properties of IgI domain I83 which is able to bind to calcium and is part of a binding site for F-actin and p94. To continue our exploration of the titin IgI domains of the N2A region we describe here the solution NMR structure of domain I82 of murine titin. The structure of the murine domain is virtually identical to the human homologue. However, the NMR investigation reveals the existence of distinct conformers around a highly conserved glycine, part of the tyrosine corner motif at the junction of the EF-loop with the F-strand. ¹⁵N relaxation data show substantial line broadening for residues around this glycine, confirming conformational exchange on the fast-intermediate time scale. The unusual dynamics could for the first time explain the high level of conservation of the tyrosine corner glycine via a function in the folding of the domain.

Monoclonal antibodies (mAbs) are essential therapeutic agents whose efficacy depends critically on the integrity of their higher-order structure (HOS). Subtle perturbations in HOS, arising from post-translational modifications, chemical degradation or formulation conditions, can impair antigen binding or induce immunogenicity. Nuclear Magnetic Resonance (NMR) spectroscopy, and particularly methyl NMR, offers a powerful means to probe the structure and dynamics of large proteins, such as antibodies, at atomic scale resolution. While 2D ¹H-¹³C methyl correlation spectra at natural abundance provide valuable structural fingerprints, their limited resolution and sensitivity hamper sophisticated NMR studies. Isotopic labeling of methyl groups with ¹³CH₃, combined with tailored deuteration, greatly enhances spectral quality. However, such labeling strategies remain underdeveloped in mammalian systems like CHO cells, which are the standard hosts for therapeutic antibody production. Here, we report an optimized labeling strategy enabling selective incorporation of the ¹³CH₃-labeled methyl groups from the six methyl-bearing amino acids into antibodies expressed in CHO cells. This includes enzymatic synthesis of regio- and stereoselectively labeled isoleucine and valine with partial deuteration and an optimized protocol for their incorporation into CHO-produced proteins. When applied to an anti-LAMP1 therapeutic antibody, this strategy yields highly resolved methyl NMR spectra, enabling comprehensive HOS assessment. It allows the transfer of previously assigned Fab and Fc methyl resonances, resulting in approximately 84% of methyl signals being assigned in the intact mAb. This work establishes a robust framework for NMR-based structural analysis of glycosylated antibodies directly from mammalian expression systems.

Over the last 20 years, the number of large proteins accessible to protein-NMR analysis has increased significantly due to the development of selective [¹H_,¹³C]-methyl labelling techniques in combination with methyl-TROSY based NMR experiments. Structure-based strategies for the assignment of [¹H,¹³C]-methyl groups rely on comparing spatial constraints derived from methyl–methyl NOEs to a known three-dimensional structure of the protein of interest. Cross peaks in methyl-TROSY spectra are assigned to specific methyl groups by matching methyl–methyl NOEs, as observed for example in 4D HMQC-NOESY-HMQC spectra, with distances derived from a structural model. This process is commonly referred to as a “methyl walk”. Here, we present AMIGO (Automated Methyl assignment via Iterative Graph Optimization), a novel assignment algorithm that formalises the intuitive methyl walk procedure by constructing graphs with nodes representing specific methyl groups and edges reflecting methyl-methyl NOEs or short methyl-methyl distances in a model. “Building blocks” consisting of nodes and edges are then generated to reconcile structure-based and NOE-based graphs. Assignments are achieved through permutation and concatenation of individual “building blocks” in a modular fashion, enabling efficient computation even for large proteins. Additional experimental restraints, such as paramagnetic relaxation enhancements (PREs) or pseudocontact shifts (PCSs), can be integrated to validate and extend the assignments. The performance of AMIGO was validated using 11 proteins that had previously been assigned and 32 NOE networks that had been generated synthetically.

Solid-state NMR spectroscopy is often limited by low spectral resolution, a problem typically addressed using fast magic-angle spinning (MAS) and ¹H detection, which require costly specialized hardware. Here, we demonstrate that the super-resolution method—previously applied in solution-state NMR—can be successfully implemented in solid-state NMR to enhance resolution. Applying dynamic number of scans (DNS) sampling to 2D ¹³C-¹³C DARR experiments on the AP205 capsid protein yielded an effective doubling of resolution, halving peak widths from ~ 180 Hz to ~ 87 Hz. Furthermore, DNS acquisition provides a significant advantage over post-acquisition apodization of conventional data with a 20% gain in sensitivity, yielding 309 more detectable peaks with 20% more sequential contacts and 25% more long-range contacts. This method is simple to implement and provides a powerful, accessible strategy to greatly improve the quality of solid-state NMR spectra applicable at all MAS frequencies.