Relaxation of protons by radicals in rotationally immobilized proteins

Proton spin-lattice relaxation by paramagnetic centers may be dramatically enhanced if the paramagnetic center is rotationally immobilized in the magnetic field. The details of the relaxation mechanism are different from those appropriate to solutions of paramagnetic relaxation agents. We report here large enhancements in the proton spin-lattice relaxation rate constants associated with organic radicals when the radical system is rigidly connected with a rotationally immobilized macromolecular matrix such as a dry protein or a cross-linked protein gel. The paramagnetic contribution to the protein-proton population is direct and distributed internally among the protein protons by efficient spin diffusion. In the case of a cross-linked-protein gel, the paramagnetic effects are carried to the water spins indirectly by chemical exchange mechanisms involving water molecule exchange with rare long-lived water molecule binding sites on the immobilized protein and proton exchange. The dramatic increase in the efficiency of spin relaxation by organic radicals compared with metal systems at low magnetic field strengths results because the electron relaxation time of the radical is orders of magnitude larger than that for metal systems. This gain in relaxation efficiency provides completely new opportunities for the design of spin-lattice relaxation based contrast agents in magnetic imaging and also provides new ways to examine intramolecular protein dynamics.     

Theory of intramolecular spin relaxation by translational diffusion in locally ordered fluids

In locally ordered fluids, such as macromolecular solutions, clays and lyotropic liquid crystals, nuclear spin relaxation can be induced by modulation, through translational diffusion of the fluid molecules, of the magnitude and orientation of the residual intramolecular spin-lattice coupling tensor, which is only partially averaged by local molecular motions near an interface. A theory of spin relaxation in locally ordered fluids bounded by planar interfaces is developed, with special emphasis on effects of translational diffusion. The theory is based on a continuous diffusion model (CDM) which, in contrast to the commonly adopted discrete exchange model (DEM), treats equilibrium and time-dependent distribution functions in a self-consistent way. A striking feature of translational diffusion in heterogeneous systems is the abundance of reencounters with previously visited interfacial regions. It is demonstrated that these diffusional reencounters, which are inherent in the CDM theory, may lead to a relaxation behaviour which is qualitatively different from that predicted by the DEM theory. Furthermore, it is seen that the widespread concept of intrinsic relaxation rate (associated with a spatial region) and the fast/slow exchange classification are not generally valid. The formal framework of the CDM theory allows molecular interactions of any complexity to be introduced. In this paper a mean-field model based on the nonlinear Poisson-Boltzmann equation is used to obtain analytic expressions for the spectral density functions that determine the relaxation behaviour in the presence and in the absence of spectral line splittings.     

Comparing continuous wave progressive saturation EPR and time domain saturation recovery EPR over the entire motional range of nitroxide spin labels

The measurement of spin-lattice relaxation rates from spin labels, such as nitroxides, in the presence and absence of spin relaxants provides information that is useful for determining biomolecular properties such as nucleic acid dynamics and the interaction of proteins with membranes. We compare X-band continuous wave (CW) and pulsed or time domain (TD) EPR methods for obtaining spin-lattice relaxation rates of spin labels across the entire range of rotational motion to which relaxation rates are sensitive. Model nitroxides and spin-labeled biological species are used to illustrate the potential complications that arise in extracting relaxation data under conditions typical to biological experiments. The effect of super hyperfine (SHF) structure is investigated for both CW and TD spectra. First and second harmonic absorption and dispersion CW spectra of the nitroxide spin label, TEMPOL, are all fit simultaneously to a model of SHF structure over a range of microwave amplitudes. The CW spectra are novel because all harmonics and microwave phases were acquired simultaneously using our homebuilt CW/TD spectrometer. The effect of the SHF structure on the pulsed free induction decay (FID) and pulsed saturation recovery spectrum is shown for both protonated and deuterated TEMPOL. We present novel pulsed saturation recovery measurements on biological molecules, including spin-lattice relaxation rates of spin-labeled proteins and spin-labeled double-stranded DNA. The impact of structure and dynamics on relaxation rates are discussed in the context of each of these examples. Collisional relaxation rates with oxygen and transition metal paramagnetic relaxants are extracted using both continuous wave and time domain methods. The extent of the errors inherent in the CW method and the advantages of pulsed methods for unambiguously measuring collisional relaxation rates are discussed. Spin-lattice relaxation rates, determined by both CW and pulsed methods, are used to determine the electrostatic potential on the surface of a protein.     

The dependence of relaxation rates and chemical shift on the size of the imaged molecules and the concentration of MRI contrast agents

An intriguing phenomenon on enhancement of the relaxation rates and chemical shift of two typical magnetic resonance imaging (MRI) contrast agents based on gadolinium complex is observed. The relaxation enhancement or chemical shift change depends on the size of the molecule where the imaged nuclear species is located: the small molecules show a perfect linear relationship between the concentration and the relaxation enhancement or chemical shift change while for macromolecules pronounced nonlinearity is observed. The phenomenon is also confirmed with real images of a macromolecular sample. A quantitative theoretical interpretation of the phenomenon is proposed and the significance of this phenomenon to MRI of materials and biological systems is discussed.     

Effects of paramagnetic cations on the nonexponential spin-lattice relaxation of rare spin nuclei in solids

Nonexponential spin-lattice relaxation is often observed for rare spin nuclei in the solid state. Deviation from single-component decay may be amplified by the coupling of rare spin nuclei to paramagnetic centers. Nonexponential spin-lattice relaxation was observed in derivatized silica gels resins. This phenomenon was localized and enhanced when paramagnetic transition metal cations were bound to surface functional groups. A stretched exponential analysis method was determined to be robust in fitting nonexponential relaxation curves for silica gels both with and without bound paramagnetic ions. Spin-lattice relaxation rates (T₁⁻¹) for functional group nuclei increased as a function of percent surface coverage with metal ion. The magnitude of the relaxation rate increase was dependent upon internuclear distances from the paramagnetic center. At low surface coverages, a semi-random distribution of paramagnetic centers increased the degree of stretching of spin-lattice relaxation decays, as measured by decreases in the calculated stretching parameter β. At higher surface coverages, calculated β values reached a limiting value, indicating that while the spin-diffusion mechanism in metal-ex-changed silica gels is restricted, it is not completely diminished.     

Soluble Spin-Label as a NMR Relaxation Probe in the Study of the Enzyme-Substrate Complexes

Abstract

The paramagnetic contributions induced by TEMPOL soluble spin-label on gentiobiose carbon spin-lattice relaxation rates are analyzed. Selective effects in the β(1–6) glycosidic bond region were observed. The possibility of using soluble spin-label to determine the stereochemistry of the substrate-enzyme interaction was then explored.

The results obtained with different diamagnetic and paramagnetic systems enabled us to distinguish the region of gentiobiose most involved in interaction with the enzyme, and the region of the disaccharide molecule located on the surface of the enzyme and most exposed to the nitroxide.

The results obtained could be used to model the enzyme surface of the gentiobiose binding site.

Soluble spin-labels have been widely used in the investigation of the conformational and dynamical properties of biomolecules in solution (1–4). The main effects of nit oxide spin-labels, like TEMPO or TEMPOL, is the increase in NMR spin-lattice relaxation rates of solvent exposed nuclei. In small molecules like the disaccharide gentiobiose, the spin-label can be used to discriminate molecular regions with a different solvent accessibility and to investigate interaction with the enzyme are the C1s, C2s and C4s carbons (where s refers to α and β and to the reducing and most reducing moiety). It is interesting to observe that although the results come from four independent systems, complementary and self consistent information was obtained on the substrate region involved in the interaction with the enzyme and on the region of the substrate in contact with the surface of the enzyme and exposed to the solvent and nitroxide molecules.

This method proved useful for investigating the surface and the conformation of the enzyme interacting with the substrate molecule, and is a new application of soluble spin label in structural studies.     

Long-lived nuclear spin states in the solution NMR of four-spin systems

The existence of long-lived nuclear spin states in four-spin systems is explored by solution-state NMR experiments. Long-lived states are proved to exist in three different natural product molecules, each containing either a AA'BB' or a AA'XX' proton spin system. The measured state lifetimes are between four and eight times the spin-lattice relaxation time constants.     

A field-cycling NMR investigation of resonant spin-lattice relaxation features arising from tunnelling methyl groups

(1)H nuclear spin-lattice relaxation has been investigated in sodium acetate trihydrate and sorbic acid using field-cycling NMR in the solid state. The relaxation is dominated by the reorientation of the methyl groups. Resonant features arising from coherent tunnelling are observed in both the magnetic field dependence of the spin lattice relaxation rate, T(1)(-1)(B(z)) and in the inverse temperature dependence, T(1)(-1)(1/T). The two systems have different barrier heights and tunnelling frequencies, providing different perspectives on the tunnel resonance phenomena. The magnetic field dependence enables different spectral density components to be separately investigated and in the carboxylic acid, sorbic acid, concerted proton transfer in the hydrogen bonds is also identified at low field and low temperature. The methyl hindering barriers and the correlation times characterising the reorientational dynamics has been accurately determined in both materials.     

Proton spin relaxation in bisphenol-a polycarbonate,butyl rubber,and their composites

Proton spin-lattice relaxation times of bisphenol-A polycarbonate, butyl rubber, and blends of the two polymers were studied at 18 Mc/sec in the temperature range 90°-450°K. The proton spin-lattice relaxation is primarily dipolar in each polymer, due to methyl group reorientation and to reorientation of chain segments. In a blend of bisphenol-A polycarbonate with 7 and 10 wt of butyl, a nonexponential decay of magnetization was observed in the temperature range 280°-380°K. This was explained by the existence of two spin temperatures in these blends, indicating that processes which bring about the equilibrium within the spin system are slow compared to the spin-lattice relaxation times of the two components of the blend.     

Dynamics of water in and around proteins characterized by 1H-spin-lattice relaxometry

Nuclear magnetic spin-lattice relaxation rate constants measured as a function of the magnetic field strength over wide ranges of Larmor frequency map the noise spectrum that drives spin relaxation. For water in and around protein systems, the spin relaxation reports on the average local translational mobility at the interface which is reduced by approximately factor of three from the bulk and there is anisotropy induced in the motions caused by the excluded volume created by the presence of the protein. Water also penetrates the protein and relatively few bound water sites provide a strong coupling between the protein dynamics and the water-proton-spin relaxation.     

Exchange and dipolar spin-spin interaction and rotational diffusion in saturation transfer EPR spectroscopy

Saturation transfer EPR spectroscopy (STEPR) provides a means for investigating weak spin-spin interaction between spin-labelled molecules because the spectral intensity is proportional to the effective spin-lattice relaxation time,T ₁ ^eff. Rate equations for the spin population defferences yield equivalent results for the dependence ofT ₁ ^eff on the physical (or chemical) and Heisenberg spin exchange rates and show thatT ₁ ^eff depends on the extent of redistribution of saturation throughout the anisotropic spin label powder lineshape. This approach yields a particularly simple formulation for the dependence of the STEPR lineshape on slow rotational diffusion. The effects of spin exchange are readily distinguished from those of slow rotational diffusion because of the insensitivity of the STEPR lineshape in the former case. The characteristic dependence of the STEPR spectral intensity on spin concentration allows determination of the exchange rate and can be used for studying slow translational diffusion, e.g. of spin-labelled proteins. Dipolar relaxation induced by paramagnetic ions gives a linear dependence of the reciprocal spin label STEPR intensity on metal ion concentration. STEPR measurements with spin-labelled lipid molecules in gel phase membranes in the presence of Ni²⁺ ions yield reliable distance information and provide calibrations for use with other systems.     

Proton NMR study of α-MnH0.06

Proton nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation rates for the solid solution α-MnH_0.06 have been measured over the temperature range 11-297 K and the resonance frequency range 20-90 MHz. A considerable shift and broadening of the proton NMR line and a sharp peak of the spin-lattice relaxation rate are observed near 130 K. These effects are attributed to the onset of antiferromagnetic ordering below the Néel temperature T_N≈130 K. The proton NMR line does not disappear in the antiferromagnetic phase; this suggests a small magnitude of the local magnetic fields at H-sites in α-MnH_0.06. The spin-lattice relaxation rate in the paramagnetic phase is dominated by the effects of spin fluctuations.     

Spin polarization in fullerene derivatives containing a nitroxide group. Observation of the intermediate photoexcited quartet state in radical triplet pair interaction

A series of C₆₀ fullerene derivatives containing a nitroxide group has been photoexcited by short LASER pulses in the microwave cavity of a cw-EPR spectrometer. Strongly spin polarized signals have been observed, in glassy matrix as well as in liquid solution, for both the ground electronic state and the excited quartet state. In the quartet state the excitation resides in the fullerene part and the molecule constitutes a triplet-radical pair with the partner covalently linked. The absorptive or emissive character of the transitions is explained in terms of the mechanism of radicaltriplet interaction producing spin polarization. Opposite initial sign and polarization patterns are observed for molecules with different spacer between nitroxide and fullerene. The time evolution of the relevant sublevel populations is fitted by a kinetic model taking into account quartet decay constants, quartet and doublet spin-lattice relaxation rates and branching ratios.     

Nuclear Relaxation Analysis of the Xenobiotic- Receptor (DNA Or Plasmatic Protein) Recognition Process

The study of interactions of xenobiotics with macromolecular receptors is very important for understanding the chemical behaviour of xenobiotic compounds in the biological organisms.

The xenobiotic molecules are able to affect the natural activities of biological receptor such as DNA or plasmatic protein. In fact the modification of the conformation of DNA or plasmatic protein, induced by interaction with xenobiotic molecules, can determine profound alterations of the normal biochemical activity.

In this study a method based on proton NMR selective and non-selective spin-lattice relaxation rate measurements and their dependence on temperature for analyzing the ability of ligand to interact with receptor is used. The NMR parameters are a weighted average between the free and bound to xenobiotic environments.     

Spin diffusion and nuclear spin-lattice relaxation in irradiated solids: a multiple-pulse NQR study

We present a detailed theoretical and experimental NQR multiple-pulse spin-locking study of spin-lattice relaxation and spin diffusion processes in the presence of paramagnetic impurities in solids. The relaxation function of the nuclear spin system at the beginning of the relaxation process is given by exp , where T_1ρ is spin-lattice relaxation time in rotating frame and =d/6, d is the sample dimensionality. Then the relaxation proceeds asymptotically to an exponential function of time, which was attributed to the spin-diffusion regime. Using the experimental data obtained from the analysis of those two relaxation regimes in γ-irradiated powdered NaClO₃, spin diffusion coefficient has been determined and the radius of the diffusion barrier has been estimated.     

The influence of macromolecular polymerization of spin-lattice relaxation of aqueous solutions

The docking or polymerization of globular proteins is demonstrated to cause changes in proton NMR spin-lattice (T1) relaxation times. Studies on solutions of lysozyme, bovine serum albumin, actin, and tubulin are used to demonstrate that two mechanisms account for the observed changes in T1. Polymerization displaces the hydration water sheath surrounding globular proteins in solution that causes an increase in T1. Polymerization also slows the average tumbling rate of the proteins, which typically causes a contrary decrease in T1. The crystallization reaction of lysozyme in sodium chloride solution further demonstrates that the "effective" molecular weight can either decrease or increase T1 depending on how much the protein is slowed. The displacement of hydration water increases T1 because it speeds up the mean motional state of water in the solution. Macromolecular docking typically decreases T1 because it slows the mean motional state of the solute molecules. Cross-relaxation between the proteins and bound water provides the mechanism that allows macromolecular motion to influence the relaxation rate of the solvent. Fast chemical exchange between bound, structured, and bulk water accounts for monoexponential spin-lattice relaxation. Thus the spin-lattice relaxation rate of water in protein solutions is a complex reflection of the motional properties of all the molecules present containing proton magnetic dipoles. It is expected, as a result, that the characteristic relaxation times of tissues will reflect the influence of polymerization changes related to cellular activities.     

Modeling magnetization transfer using a three-pool model and physically meaningful constraints on the fitting parameters.

A model for water-macromolecular magnetization transfer is presented which addresses the mechanism of coupling between the hydrogen populations and the extraction of physically meaningful parameters from experimental magnetization transfer data. Both physical exchange between bulk-solvent and site-specific hydration-layer hydrogens and intermolecular magnetic dipolar coupling between these specific hydration-layer-solvent and macromolecular hydrogens are explicitly included, leading to a three-pool model for magnetization transfer. It is shown that the three-pool model is well approximated by a two-pool model for coupling between the bulk-solvent and macromolecular hydrogens when the dipolar-coupled solvent hydrogens are a small fraction of the total solvent, and the solvent-macromolecular coupling constant includes both dipolar magnetic, kappa(dip), and physical exchange, kappa(ex), coupling rates. The model is also extended to multiple solvent systems. The model results in a set of coupled equations that predict magnetization transfer spectra as a function of temperature and composition. Physically meaningful constraints on the coupling and relaxation parameters are established for systems in which magnetization transfer has been observed including solvated cross-linked proteins and lipid bilayers. Using parameter estimates based on these constraints, empirical magnetization transfer spectra are well predicted by the model. It is found that the degree of magnetization transfer becomes independent of kappa(dip) and kappa(ex) when these parameters become greater than about 50 s(-1). In the semi-rigid cross-linked protein systems where the mobility of the macromolecular matrix is insensitive to temperature, the magnitude of the observed magnetization transfer is consistent with being limited by the intermolecular dipolar coupling and spin-lattice relaxation in the bulk-solvent phase.     

Spin-lattice relaxation in hyperfine-coupled systems: Applications to interstitial diffusion and molecular dynamics

The solid state diffusion of hydrogen, or of its pseudo-isotope muonium, provides an interesting example of spin-lattice relaxation in a 2-spin, 4-level system. The local field experienced by the interstitial atom fluctuates as it moves, inducing transitions between the coupled electron and nuclear spin states. Rate equations governing the populations of these states may be solved numerically to simulate the different relaxation functions which would be displayed by ESR, ENDOR and μSR spectroscopies and to assist in extracting motional correlation times from the experimental data. Spin relaxation in molecular radicals may be treated similarly, with different selection rules for different mechanisms: this paper treats the spin rotation mechanism and perturbation to anisotropic or isotropic components of the hyperfine interaction, caused by inter or intra-molecular motion. Conventional magnetic resonance monitors the population differences appropriate to particular transitions; only in sufficiently high fields do these distinguish the electronic and nuclear response. Muon spin relaxation is remarkable in separating out the nuclear spin projection whatever the degree of mixing of the spin states,via the asymmetry in the muon radioactive decay. Experimentally it has the advantage that measurements can be made over a wide range of field, from null external field up to thelevel crossing where the relaxation rate exhibits a striking peak.     

Zeeman effect in complex molecules in solid solutions with laser T1 → S0 excitation. New possibilities for investigation of spin—lattice relaxation

The main peculiarities of Zeeman-effect in phosphorescence spectra of randomly oriented molecular systems under selective T₁→S₀ excitation are investigated. On this basis a new method of measuring triplet state characteristics and spin—lattice relaxation rates in solid solutions is proposed. The results of Zeeman-effect investigations on 5-bromo-acenaphtene in ethanol and butylbromide at magnetic fields 10–50 kG at helium temperatures are given as an example. At low fields the deviation from Boltzmann equilibrium in the spin system of the molecule is observed and the rates of spin—lattice relaxation are determined.     

Spin-wave description of nuclear spin-lattice relaxation in Mn12O12 acetate

In response to recent nuclear-magnetic-resonance (NMR) measurements on the molecular cluster Mn12O12 acetate, we study the nuclear spin-lattice relaxation rate 1/T(1), developing a modified spin-wave theory. Our microscopic new approach, which is distinct from previous macroscopic treatments of the cluster as a rigid spin of S=10, not only excellently interprets the observed temperature and applied-field dependences of 1/T(1) for 55Mn nuclei but also strongly supports the 13C NMR evidence for spin delocalization over the entire molecule.