Communication

Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins

Intrinsically disordered proteins (IDPs) defy the conventional structure-function paradigm by lacking a well-defined tertiary structure and exhibiting inherent flexibility. This flexibility leads to distinctive spin relaxation modes, reflecting isolated and specific motions within individual peptide planes. In this work, we propose a new pulse sequence to measure the longitudinal ¹³C${}^{\prime }$ CSA-¹³C${}^{\prime }$-¹³C${}^{\alpha }$ DD CCR rate ${\Gamma }_z^{C^{\prime }/C^{\prime }{C}^{\alpha }}$ and present a novel 3D version of the transverse ${\Gamma }_{xy}^{C^{\prime }/C^{\prime }{C}^{\alpha }}$ CCR rate, adopting the symmetrical reconversion approach. We combined these rates with the analogous ${\Gamma }_{xy}^{N/NH}$ and ${\Gamma }_z^{N/NH}$ CCR rates to derive residue-specific correlation times for both spin-pairs within the same peptide plane. The presented approach offers a straightforward and intuitive way to compare the correlation times of two different and complementary spin vectors, anticipated to be a valuable aid to determine IDPs backbone dihedral angles distributions. We performed the proposed experiments on two systems: a folded protein ubiquitin and Coturnix japonica osteopontin, a prototypical IDP. Comparative analyses of the results show that the correlation times of different residues vary more for IDPs than globular proteins, indicating that the dynamics of IDPs is largely heterogeneous and dominated by local fluctuations.

Biomolecular solid-state magic angle spinning (MAS) NMR spectroscopy frequently relies on selective ¹³C-¹⁵N magnetization transfers, for various kinds of correlation experiments. Introduced in 1998, spectrally induced filtering in combination with cross polarization (SPECIFIC-CP) is a selective heteronuclear magnetization transfer experiment widely used for biological applications. At MAS frequencies below 20 kHz, commonly used for ¹³C-detected MAS NMR experiments, SPECIFIC-CP transfer between amide ¹⁵N and ¹³C^α atoms (NCA) is typically performed with radiofrequency (rf) fields set higher than the MAS frequency for both ¹³C and ¹⁵N channels, and high-power ¹H decoupling rf field is simultaneously applied. Here, we experimentally explore a broad range of NCA zero-quantum (ZQ) SPECIFIC-CP matching conditions at the MAS frequency of 14 kHz and compare the best high- and low-power matching conditions with respect to selectivity, robustness, and sensitivity at lower ¹H decoupling rf fields. We show that low-power NCA SPECIFIC-CP matching condition gives rise to 20% sensitivity enhancement compared to high-power conditions, in 2D NCA spectra of microcrystalline assemblies of HIV-1 CA_CTD-SP1 protein with inositol hexakis-phosphate (IP6).

An NMR experiment is designed for accurate and robust measurement of transverse relaxation rates of degenerate ¹H transitions in selectively ¹³CH₃-labeled, deuterated small proteins. The measurement is based on the use of acute (<90°) angle ¹H radio-frequency pulses and relies on selection of the slow- and fast-relaxing components of methyl magnetization following the relaxation period in separate experiments. The R₂ decay series recorded with selection of the fast-relaxing components serves as a useful complement to the R₂ series acquired with selection of the slow-relaxing part, and permits the extension of the range of relative contributions of the fast- and slow-relaxing parts to apparent signal decay. The approach is experimentally verified on ¹³CH₃ methyl groups of the ILV-{¹³CH₃}-labeled protein ubiquitin at 10 °C and 25 °C. The obtained methyl ¹H relaxation rates are in remarkably good agreement with the values obtained from well-established NMR techniques.

3D iDOSY-HMBC (3D incorporated Diffusion Ordered SpectroscopY-Heteronuclear Multiple Bond Correlation) pulse sequences were modified to incorporate convection compensation element. No additional delays were required, and convection compensation was directly constructed within the existing delay periods in 3D iDOSY-HMBC pulse sequence. Convection compensation was achieved by pulsed field gradient double echo, thus avoiding the intensity loss normally related to stimulated echo methods. The incorporated convection compensation element improves the usability of 3D iDOSY-HMBC. In case of elevated temperatures, thermal convection leads to loss of resolution in DOSY-dimension or even to severe decrease of signal intensity, thus making convection uncompensated 3D iDOSY-HMBC infeasible. Aforementioned problems can be circumvented via utilization of the presented convection compensated 3D iDOSY-HMBC-versions.

Over 40% of eukaryotic proteomes and 15% of bacterial proteomes are predicted to be intrinsically disordered based on their amino acid sequence. Intrinsically disordered proteins (IDPs) exist as heterogeneous ensembles of interconverting conformations and pose a challenge to the structure–function paradigm by apparently functioning without possessing stable structural elements. IDPs play a prominent role in biological processes involving extensive intermolecular interaction networks and their inherently dynamic nature facilitates their promiscuous interaction with multiple structurally diverse partner molecules. NMR spectroscopy has made pivotal contributions to our understanding of IDPs because of its unique ability to characterize heterogeneity at atomic resolution. NMR methods such as Chemical Exchange Saturation Transfer (CEST) and relaxation dispersion have enabled the detection of ‘invisible’ excited states in biomolecules which are transiently and sparsely populated, yet central for function. Here, we develop a ¹Hα CEST pulse sequence which overcomes the resonance overlap problem in the ¹Hα-¹³Cα plane of IDPs by taking advantage of the superior resolution in the ¹H-¹⁵N correlation spectrum. In this sequence, magnetization is transferred after ¹H CEST using a triple resonance coherence transfer pathway from ¹Hα (i) to ¹HN(i + 1) during which the ¹⁵N(t₁) and ¹HN(t₂) are frequency labelled. This approach is integrated with spin state-selective CEST for eliminating spurious dips in CEST profiles resulting from dipolar cross-relaxation. We apply this sequence to determine the excited state ¹Hα chemical shifts of the intrinsically disordered DNA binding domain (CytR^N) of the bacterial cytidine repressor (CytR), which transiently acquires a functional globally folded conformation. The structure of the excited state, calculated using ¹Hα chemical shifts in conjunction with other excited state NMR restraints, is a three-helix bundle incorporating a helix-turn-helix motif that is vital for binding DNA.

This study investigates the bioactivity and microstructural properties of borate-based bioactive 13-93B3 glass produced by the solution combustion synthesis (SCS) method using urea and sucrose fuels at different reaction temperatures. The results show these glasses exhibit unique properties compared to conventionally-synthesized borate-based bioactive glasses. The glass particles produced have porous surfaces, with particle sizes below 100 nm. BET measurements reveal that the mesoporous structure of these glasses is characterized by high-specific surface areas, promoting a high ion release and hydroxyapatite layer formation in simulated body fluid (SBF). The crystallization of the amorphous calcium phosphate was investigated by ¹H, ³¹P MAS, and 2D ³¹P{1H} heteronuclear correlation (HETCOR) spectroscopy, showing that it progresses linearly up to three days. The produced glasses were tested for their effects on the viability of 3T3 cells and were found to have no toxic effects. Therefore, the produced glasses are promising candidates for tissue engineering applications.