Journal of Biomolecular NMR最新文献

Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand–protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous ¹H-¹⁵?N, ¹H-¹³C SESAME based pulse scheme for the rapid acquisition of ¹H^C/N-R₂ relaxation rates for the determination of backbone and sidechain PREs of proteins. The ¹H^N-R₂ rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R²?=?0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.

In-cell NMR spectroscopy provides precious structural and functional information on biological macromolecules in their native cellular environment at atomic resolution. However, the intrinsic low sensitivity of NMR imposes a big limitation in the applicability of the methodology. In this respect, the recently developed commercial 1.2?GHz NMR spectrometer is expected to introduce significant benefits. However, cell samples may suffer from detrimental effects at ultrahigh fields, that must be carefully evaluated. Here we show the first in-cell NMR spectra recorded at 1.2?GHz on human cells, and we compare resolution and sensitivity against those obtained at 900 and 950?MHz. To evaluate the effects of different spin relaxation rates, SOFAST-HMQC and BEST-TROSY spectra were recorded on intracellular α-synuclein and carbonic anhydrase. Major improvements are observed at 1.2?GHz when analyzing unfolded proteins, such as α-synuclein, while the TROSY scheme improves the resolution for both globular and unfolded proteins.

A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val ¹H/¹³C methyl protonated proteins from 100?ml cultures in M9?++?/D₂O medium induced at high (OD₆₀₀?~?10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of ²H/¹⁵N/¹³C and ¹H/¹³C labeled proteins, yields comparable quantities of protein from 100?ml cell culture to those obtained using a conventional 1 L culture with M9/D₂O medium, while using three-fold less α-ketoisovaleric (1,2,3,4-¹³C₄; 3,4′,4′,4′-d₄) and α-ketobutyric (¹³C₄; 3,3-d₂) acid precursors.

G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in E. coli has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR E. coli expression and then describe the development of an optimized robust protocol for the E. coli expression and purification of two mutants of the turkey β₁-adrenergic receptor (β₁AR) uniformly or selectively labeled in ¹⁵N or ²H,¹⁵N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for E. coli expression. Optimization of E. coli expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2–0.3?mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-β₁AR mutant also comprises the two native tyrosines Y^5.58 and Y^7.53, which enable G protein binding. High-quality ¹H-¹⁵N TROSY spectra were obtained for E. coli-expressed YY-β₁AR in three different functional states (antagonist, agonist, and agonist?+?G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.

Values of ³J-couplings as obtained from NMR experiments on proteins cannot easily be used to determine protein structure due to the difficulty of accounting for the high sensitivity of intermediate ³J-coupling values (4–8?Hz) to the averaging period that must cover the conformational variability of the torsional angle related to the ³J-coupling, and due to the difficulty of handling the multiple-valued character of the inverse Karplus relation between torsional angle and ³J-coupling. Both problems can be solved by using ³J-coupling time-averaging local-elevation restraining MD simulation. Application to the protein hen egg white lysozyme using 213 backbone and side-chain ³J-coupling restraints shows that a conformational ensemble compatible with the experimental data can be obtained using this technique, and that accounting for averaging and the ability of the algorithm to escape from local minima for the torsional angle induced by the Karplus relation, are essential for a comprehensive use of ³J-coupling data in protein structure determination.

The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of?+?18?Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D?NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pK_a values of isoAsp and C-terminal Asp in short peptides. The characteristic ¹H-¹³C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4–5) and 40?°C. The results show that the application of our 2D?NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.

Advanced NMR methods combined with biophysical techniques have recently provided unprecedented insight into structure and dynamics of molecular chaperones and their interaction with client proteins. These studies showed that several molecular chaperones are able to dissolve aggregation-prone polypeptides in aqueous solution. Furthermore, chaperone-bound clients often feature fluid-like backbone dynamics and chaperones have a denaturing effect on clients. Interestingly, these effects that chaperones have on client proteins resemble the effects of known chaotropic substances. Following this analogy, chaotropicity could be a fruitful concept to describe, quantify and rationalize molecular chaperone function. In addition, the observations raise the possibility that at least some molecular chaperones might share functional similarities with chaotropes. We discuss these concepts and outline future research in this direction.

Unidirectional coherence transfer is highly efficient in intrinsically disordered proteins (IDPs). Their elevated ps-ns timescale dynamics ensures long transverse (T₂) relaxation times allowing sophisticated coherence transfer pathway selection in comparison to folded proteins. ¹H^α-detection ensures non-susceptibility to chemical exchange with the solvent and enables chemical shift assignment of consecutive proline residues, typically abundant in IDPs. However, many IDPs undergo a disorder-to-order transition upon interaction with their target protein, which leads to the loss of the favorable relaxation properties. Long coherence transfer routes now result in prohibitively large decrease in sensitivity. We introduce a novel 4D ¹H^α-detected experiment HACANCOi, together with its 3D implementation, which warrant high sensitivity for the assignment of proline-rich regions in IDPs in complex with a globular protein. The experiment correlates ¹H^α_i, ¹³C^α_i, ¹⁵N_i and (^{13} C^{prime}_{i}) spins by transferring the magnetization concomitantly from ¹³C^α_i to ¹⁵N_i and (^{13} C^{prime}_{i}). The B1 domain of protein G (GB1), and the enteropathogenic E. coli EspF in complex with human SNX9 SH3, serve as model systems to demonstrate the attainable sensitivity and successful sequential assignment.