Four-dimensional NOE-NOE spectroscopy of SARS-CoV-2 Main Protease to facilitate resonance assignment and structural analysis

Resonance assignment and structural studies of larger proteins by nuclear magnetic resonance (NMR) can be challenging when exchange broadening, multiple stable conformations, and 1H back-exchange of the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2 × 306 residues. We demonstrate that the combination of four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy and 4D NOESY-NOESY-TROSY spectroscopy provides an effective tool for delineating the 1H–1H dipolar relaxation network. In combination with detailed structural information obtained from prior X-ray crystallography work, such data are particularly useful for extending and validating resonance assignments as well as for probing structural features.

Four-dimensional NOE-NOE spectroscopy of SARS-CoV-2 Main Protease to facilitate resonance assignment and structural analysis

Resonance assignment and structural studies of larger proteins by NMR can be challenging when exchange broadening, multiple stable conformations, and back-exchanging the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2×306 residues. We demonstrate that the combination of four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy and 4D NOESY-NOESY-TROSY spectroscopy provides an effective tool for delineating the 1H-1H dipolar relaxation network. In combination with detailed structural information obtained from prior X-ray crystallography work, such data are particularly useful for extending and validating resonances assignments, as well as for probing structural features.

Protein dynamics insights from ¹⁵N-¹H (TROSY) HSQC

Protein dynamic information is customarily extracted from 15N NMR spin-relaxation experiments. These experiments can only be applied to (small) proteins that can be dissolved to high concentrations. However, most proteins of interest to the biochemical and biomedical community are large and relatively insoluble. These proteins often have functional conformational changes, and it is particularly regretful that these processes cannot be supplemented by dynamical information from NMR. We ask here whether (some) dynamic information can be obtained form the 1H line widths in 15N-1H HSQC spectra. Such spectra are widely available, also for larger proteins. We developed a computer program to predict amide proton line widths from (crystal) structures. As a calibration, we test our approach on BPTI. We find that we can predict most of the distribution of experimental amide proton line widths if we take the dipole-dipole interaction with at least 40 surrounding protons into account. When focusing our attention the outliers of the distribution, we find for BPTI a cluster of conformationally broadened 1HN resonances of residues in strands 10–15 and 36–40 of the beta sheet. Conformational exchange broadening of the 15NH resonances for these residues was previously reported using 15N relaxation measurements (Szyperski et al., J. Biomol. NMR 3, 151–164, 1993). There is little or no evidence for motional narrowing of the 1HN resonances, also in agreement with earlier data using 15N relaxation methods (Beeser et.al, J. Mol. Biol. 269, 154–164, 1997). We also apply our program to 42 kDa domain of the human Hsc70 protein. In this case, there is no previous 15N relaxation data to compare with, but we find, again from the outliers of the distribution, both exchange broadening and motional narrowing that appears to corroborate previous conformational insights for this domain.

Спин-обменное уширение магнитного M-=SUB=-X-=/SUB=--резонанса в цезии

The paper presents the results of measuring the cross section of the spin-exchange broadening of the magnetic resonance line in the Zeeman structure of the ground state of cesium. The analysis of the influence of measurement errors of parameters on the result was carried out, the values of the concentration of cesium vapors were refined by independent measurements.

Методы возбуждения параметрического резонанса в схеме оптического магнитометрического датчика

An experimental comparison of the methods for modulating the parameters of resonant transverse to the external magnetic field pumping radiation in a two-beam optical magnetometric sensor (so called Bell-Bloom scheme) is carried out, as well as a comparison of these methods with the standard method of radio-frequency excitation of magnetic resonance under conditions of strong laser pumping. It is shown that although the standard method allows one to achieve a greater suppression of the spin-exchange broadening of the magnetic resonance line by pumping light, the Bell-Bloom scheme has advantages that make it possible to obtain similar sensitivity values upon modulation of both the intensity and polarization of the pump light; at the same time, the Bell-Bloom scheme is potentially characterized by higher speed, which is essential for the problems of magnetoencephalography and ultra-low field magnetic resonance imaging.

Трехуровневое приближение при расчете параметров оптически детектируемого магнитного резонанса в условиях сильной лазерной накачки

An algorithm is proposed for the approximate solution of a purely nonlinear problem of the parameters of optically detected magnetic resonance in the ground state of alkali atoms in an optically dense medium under conditions of intense narrow-band optical pumping, which causes, first, bleaching of the atomic medium, and, second, partial suppression of spin-exchange broadening. The direct solution of the Liouville equation under these conditions is complicated by the fact that the relaxation time of each of the levels of the hyperfine and Zeeman structure of the ground state is determined by the populations of the remaining levels, which leads to the necessity of solving a self-consistent problem in a multilevel system, and, as a rule, requires the use of supercomputers. In this paper, approximations are proposed that make it possible to significantly simplify and speed up the calculation by orders of magnitude, and the results are compared with experimental data using the example of a two-beam Mx scheme of a magnetometric sensor

Detection of Chemical Exchange in Methyl Groups of Macromolecules

<div> <div> <div> <p>The zero- and double-quantum methyl TROSY Hahn-echo and the methyl ¹H-¹H dipole- dipole cross-correlation nuclear magnetic resonance experiments enable estimation of multiple quantum chemical exchange broadening in methyl groups in proteins. The two relaxation rate constants are established to be linearly dependent using molecular dynamics simulations and empirical analysis of experimental data. This relationship allows chemical exchange broadening to be recognized as an increase in the Hahn-echo relaxation rate constant. The approach is illustrated by analyzing relaxation data collected at three temperatures for <i>E. coli </i>ribonuclease HI and by analyzing relaxation data collected for different cofactor and substrate complexes of <i>E. coli </i>AlkB. </p> </div> </div> </div>

Detection of Chemical Exchange in Methyl Groups of Macromolecules

<div> <div> <div> <p>The zero- and double-quantum methyl TROSY Hahn-echo and the methyl ¹H-¹H dipole- dipole cross-correlation nuclear magnetic resonance experiments enable estimation of multiple quantum chemical exchange broadening in methyl groups in proteins. The two relaxation rate constants are established to be linearly dependent using molecular dynamics simulations and empirical analysis of experimental data. This relationship allows chemical exchange broadening to be recognized as an increase in the Hahn-echo relaxation rate constant. The approach is illustrated by analyzing relaxation data collected at three temperatures for <i>E. coli </i>ribonuclease HI and by analyzing relaxation data collected for different cofactor and substrate complexes of <i>E. coli </i>AlkB. </p> </div> </div> </div>