Analysis of laboratory-frame and rotating-frame cross-relaxation buildup rates from macromolecules (original) (raw)
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Consequences of magnetization transfer on the determination of solution structures of proteins
Journal of Magnetic Resonance (1969), 1989
The complete two-dimensional nuclear Overhauser enhancement (NOESY) spectra of several proteins were theoretically simulated to analyze the effect that spin diffusion has on the cross-peak intensity-interproton distance relationship as a function of rotational time T and mixing time t,. The NOESY cross-peak intensities were calculated by solving the generalized Bloch equations from the crystal coordinates of basic pancreatic trypsin inhibitor, cytochrome b562, domain 1 of phage lysozyme, and human deoxy hemoglobin (MW 6500, 11,459, 18,625, and 64,450, respectively). It was found that correlations between cross-peak intensity and internuclear distance, useful for obtaining solution structure of proteins from nuclear magnetic resonance data, can be defined for cross peaks observed at t, = 100 ms, for rotational correlation times T < 2.3 ns (corresponding to MW < 11,459 at 68"C), and at t, = 50 ms, for r < 5.9 ns (corresponding to MW < 30,000 at 68°C). Above these limits, the correlations start to break down, and two protons at any given distance may give rise to NOE cross peaks varying greatly in intensity, depending on the local structure of the protein. o 1989 Academic FWSS. hc.
Insight into protein dynamics from nuclear magnetic relaxation studies
Polimery, 2007
In the review (63 references) the nuclear magnetic relaxation which is a unique experimental method giving insight into dynamic processes existing in proteins and covering a broad range of time scales was presented. This method, however, is demanding experimentally and theoretically. Exprimental methods limited to 15 N nuclei are briefly presented and their limitations discussed. Analysis of experimental relaxation data for proteins can be done in the frame of model-free approach or applying spectral density mapping. Both those approaches are difficult for the physical interpretation of results. Besides motional parameters, some structural parameters influence relaxation rates and have to be estimated or determined. Hopefully, many problems connected with the analysis of relaxation data in proteins can be overcome with relaxation measurements at multiple magnetic fields for different isotopes like 15 N, 13 C, and 2 H.
Protein dynamics by ¹⁵N nuclear magnetic relaxation
Methods in molecular biology (Clifton, N.J.), 2012
Nitrogen-15 relaxation is the most ubiquitous source of information about protein (backbone) dynamics used by NMR spectroscopists. It provides the general characteristics of hydrodynamics as well as internal motions on subnanosecond, micro- and millisecond timescales of a biomolecule. Here, we present a full protocol to perform and analyze a series of experiments to measure the (15)N longitudinal relaxation rate, the (15)N transverse relaxation rate under an echo train or a single echo, the (15)N-(1)H dipolar cross-relaxation rate, as well as the longitudinal and transverse cross-relaxation rates due to the cross-correlation of the nitrogen-15 chemical shift anisotropy and the dipolar coupling with the adjacent proton. These rates can be employed to carry out model-free analyses and can be used to quantify accurately the contribution of chemical exchange to transverse relaxation.
Biophysical Journal, 1999
The distance dependence of electron-nuclear dipole-dipole coupling was tested using a series of poly-Lproline based peptides of different length. The poly-proline based peptides were synthesized with a nitroxide spin label on the N-terminus and a tryptophan on the C-terminus, and paramagnetic enhancements of nuclear spin-lattice relaxation rates were measured for the aromatic protons on the tryptophan as a function of the number of proline spacers in the sequence. As expected, paramagnetic enhancements decrease with distance, but the distances deduced from the NMR relaxation rates were shorter than expected for every peptide studied compared to a rigid linear poly-L-proline type II helix structure. Calculations of cross-relaxation rates indicate that this difference is not the result of spin-diffusion or the creation of a spin-temperature gradient in the proton spins caused by the nitroxide. Molecular dynamics simulations were used to estimate dynamically averaged value of ͗r Ϫ3 ͘ 2. These weighted average distances were close to the experimentally determined distances, and suggest that molecular motion may account for differences between the rigid linear models and the distances implied by the NMR relaxation data. A poly-L-prolone peptide synthesized with a central glycine hinge showed dramatic relaxation rate enhancements compared to the peptide of the same length lacking the hinge. Molecular dynamics simulations for the hinged peptide support the notion that the NMR data is a representation of the weighted average distance, which in this case is much shorter than that expected for an extended conformation. These results demonstrate that intermoment distances based on NMR relaxation rates provide a sensitive indicator of intramolecular motions.
Journal of biomolecular NMR, 2018
Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein-protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes (N or C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model protein-protein complexes, making this approach time consuming and expensive. Here, we show a new strategy that combines a modified pulse sequence (H-Γ-CCLS) with an asymmetric labeling scheme to enable the detection of both intra- and inter-molecular PREs simultaneously using only one sample preparation. We applied this strategy to the non-covalent dimer of ubiquitin. Our method confirmed the previously identified binding interface for the transient di-ubiquitin complex, and at the same time,...
Journal of the American Chemical Society, 2007
Since the recent availability of high sensitivity field-cycling relaxometers, it has become possible to measure the protein proton relaxation in millimolar protein solutions as a function of magnetic field. In principle, this provides direct access to the so-called spectral density function of protein protons and, hence, to a full set of dynamic parameters. Understanding the dynamic behavior of biological molecules is increasingly appreciated as crucial to understanding their function. However, theoretical tools to analyze the collective relaxation behavior of protons in solute macromolecules over a wide range of magnetic fields are lacking. A complete relaxation matrix analysis of such behavior is described here. This analysis provides excellent predictions of the experimental proton magnetization decays/recoveriessmeasured to an unprecedented level of accuracy by a last-generation fast field-cycling relaxometersof two different globular proteins, hen egg white lysozyme and human serum albumin. The new experimentally validated theoretical model is then used to extract dynamic information on these systems. A "collective" order parameter SC 2 , different from, but complementary to, that commonly extracted from heteronuclear relaxation measurements at high field, is defined and measured. An accurate estimate of the rotational correlation time is obtained: in the case of lysozyme it agrees very well with theoretical predictions; in the case of serum albumin it provides evidence for aggregation at millimolar concentration.
Protein-small cosolute molecule interactions are ubiquitous and known to modulate the solubility, stability, and function of many proteins. Characterization of such transient weak interactions at atomic resolution remains challenging. In this work, we develop a simple and practical NMR method for extracting both energetic and dynamic information on protein-cosolute interactions from solvent paramagnetic relaxation enhancement (sPRE) measurements. Our procedure is based on an approximate (non-Lorentzian) spectral density that behaves exactly at both high and low frequencies. This spectral density contains two parameters, one global related to the translational diffusion coefficient of the paramagnetic cosolute, and the other residue specific. These parameters can be readily determined from sPRE data, and then used to calculate analytically a concentration normalized equilibrium average of the interspin distance, ⟨r −6 ⟩ norm , and an effective correlation time, τ C , that provide measures of the energetics and dynamics of the interaction at atomic resolution. We compare our approach with existing ones, and demonstrate the utility of our method using experimental 1 H longitudinal and transverse sPRE data recorded on the protein ubiquitin in the presence of two different nitroxide radical cosolutes, at multiple static magnetic fields. The approach for analyzing sPRE data outlined here provides a powerful tool for deepening our understanding of extremely weak protein-cosolute interactions.
Journal of Magnetic Resonance (1969), 1990
A novel method is proposed for quantifying two-dimensional, phase-sensitive, crossrelaxation spectra of proteins. Relative cross-peak volumes are calculated from peak heights and linewidths (measured along the x and y axes). We show that this method gives the same result for isolated peaks as direct volume integration. In the case of moderate peak overlap, our method is less prone to error than volume integration. Computerization of the method is easily implemented and can be used for measuring the massive sets of cross-peak volumes contained in a series of two-dimensional NMR spectra. This approach has enabled us to use a quadratic approximation to the initial build-up rates to determine cross-relaxation rates for 90 proton pairs in a protein, in the laboratory and rotating frames of reference (J. Fejzo, Zs. Zolnai, S. Macura, and J. L. Markley, J. Magn. Reson. 82, 5 18, 1989). The molecule studied was turkey ovomucoid third domain (OMTKY3), a small (6.1 kDa) globular protein.