Biomedical subjects

Robert G Bryant

Publications and source records attributed to Robert G Bryant.

16 recordsLinked to original sources

PubMed2006

Structural and dynamical examination of the low-temperature glass transition in serum albumin.

The nuclear magnetic transverse decay and the proton second moment of bovine serum albumin samples dry and hydrated with different water isotope compositions show that at temperatures around 170 K, there is a dramatic change in the dynamics of the water associated with the protein interface. By comparison, observation of the protein protons when hydrated with deuterium oxide provides no evidence for significant dynamical changes near 170 K. The proton second moment of the hydrated protein shows that the protein structure becomes more open with increasing hydration from the lyophilized condition and that the side chains extend from the protein surface into the solvent in the hydrated but not the dry cases. The proton second moment of serum albumin hydrated with H(2)O increases dramatically with decreasing temperature near 170 K, demonstrating that the water forms a rigid solid around the protein which effectively fills the surface irregularities created by the protein fold. Solvation with dimethyl sulfoxide yields small effects compared with water.

Computer Simulation↗

PubMed2006

Paramagnetic relaxation of protons in rotationally immobilized proteins.

The proton magnetic relaxation dispersion profiles are reported over the proton Larmor frequency range from 0.01 to 30 MHz for cross-linked gels and for the dry lyophilized bovine serum albumin covalently labeled at lysine with diethylenetriaminepentaacetic acid chelates of either Gd(III) or Mn(II) ions. The proton spin-lattice relaxation dispersion for the cross-linked paramagnetic protein gel is accurately represented as a sum of two major relaxation contributions. The diamagnetic term is a power law from the magnetic field dependence of the protein protons. The paramagnetic term is approximately described by the Solomon-Bloembergen-Morgan class of models. However, the paramagnetic relaxation mechanism in the dry lyophilized protein is fundamentally different and we develop a new quantitative description of the dispersion profile. In the dry case, no peak in the proton relaxation dispersion profile is detected from the field dependence of the electron spin relaxation times. The high-field paramagnetic relaxation dispersion is a power law in the Larmor frequency with an exponent of -0.8, which results from modulation of the electron-nuclear coupling by the intramolecular dynamics of the protein which primarily propagates along the primary structure of the protein. The low-field plateau is caused by the interruption of the electron-nuclear spin correlation by electron spin relaxation. This new quantitative description provides a simple approach to the measurement of electron spin-lattice relaxation times in paramagnetic protein systems at room temperature based on the magnetic field dependence of the proton spin-lattice relaxation rate constant.

Electron Spin Resonance Spectroscopy↗

PubMed2006

Oxygen accessibility to ribonuclease a: quantitative interpretation of nuclear spin relaxation induced by a freely diffusing paramagnet.

The nuclear spin relaxation induced by a freely diffusing paramagnetic center provides a direct measure of intermolecular accessibility. A number of factors are involved in a quantitative interpretation of relaxation data including excluded volume effects, solvation differences, and the details of the electron spin relaxation in the paramagnetic center. In the case where the electron relaxation time is short compared with correlation times describing the electron-nuclear coupling, the nuclear spin relaxation rates may be related to the effective local concentration of the paramagnetic center at different locations about the solute of interest. The local concentrations may in turn be related to differences in the local free energies of interaction between the diffusing paramagnet and the cosolute. We demonstrate this approach for the case of ribonuclease A and deduce surface free energy differences for a large number of protein proton sites. We find that the oxygen accessibility is poorly represented by hard-sphere models such as computed solvent or steric accessibility. There is a distribution of local intermolecular interactions with a width of the order of RT that dominates the report of the intermolecular exploration of the protein by this simple solute.

Electron Spin Resonance Spectroscopy↗

PubMed2005

Spin relaxation measurements of electrostatic bias in intermolecular exploration.

We utilize the paramagnetic contribution to proton spin-lattice relaxation rate constants induced by freely diffusing charged paramagnetic centers to investigate the effect of charge on the intermolecular exploration of a protein by the small molecule. The proton NMR spectrum provided 255 resolved resonances that report how the explorer molecule local concentration varies with position on the surface. The measurements integrate over local dielectric constant variations, and, in principle, provide an experimental characterization of the surface free energy sampling biases introduced by the charge distribution on the protein. The experimental results for ribonuclease A obtained using positive, neutral, and negatively charged small nitroxide radicals are qualitatively similar to those expected from electrostatic calculations. However, while systematic electrostatic trends are apparent, the three different combinations of the data sets do not yield internally consistent values for the electrostatic contribution to the intermolecular free energy. We attribute this failure to the weakness of the electrostatic sampling bias for charged nitroxides in water and local variations in effective translational diffusion constant at the water-protein interface, which enters the nuclear spin relaxation equations for the nitroxide-proton dipolar coupling.

Journal Article↗

PubMed2005

The aqueous reference for ESR oximetry.

The interaction of molecular oxygen with derivatives of nitroxide EPR spin labels has been investigated using nuclear spin-relaxation spectroscopy in aqueous and nonaqueous solvents. The proton spin-lattice relaxation rate induced by oxygen provides a measure of the local concentration of oxygen, which we find is dependent on solvent. In water, the hydrophobic effect increases the local concentration of oxygen in the nonpolar portions of solute molecules. For nitroxides reduced to the hydroxylamine in aqueous solutions, we find that the local concentration of oxygen is approximately twice that associated with a free diffusion hard sphere limit, while in octane, this effect is absent. These results show that nitroxide based ESR oximetry may suffer a reference concentration shift of order a factor of two if the aqueous nitroxide spectrum or relaxation is used as the reference.

Chloroform↗

PubMed2005

Noise and functional protein dynamics.

The magnetic field dependence of the proton-spin-lattice relaxation rate in rotationally immobilized proteins shows that the one-dimensional character of the protein primary structure causes a dramatic increase in the population of low-frequency motions from 10 kHz to 20 MHz. As a consequence, the probability and rate at which functionally critical conformational states are thermally sampled in a protein are dramatically increased as well, when compared with a three-dimensional lattice structure. Studies of protein dynamics often focus on time periods far shorter than those associated with catalytic function, but we show here that the magnetic field dependence of the proton nuclear spin-lattice relaxation rate in rotationally immobilized proteins reports unambiguously the structural fluctuations in the frequency range from 10 kHz to 20 MHz. This relaxation rate decreases with increasing Larmor frequency according to a power law that derives from the distribution of dynamical states, the localization of the structural disturbances, and the spatial distribution of hydrogen atoms in the structure. The robust theoretical foundation for the spin-relaxation process, loosely characterized as a direct spin-phonon coupling, shows that the disturbances propagate in a space of reduced dimensionality, essentially along the stiff connections of the polypeptide chain. The reduced dimensionality traps the disturbance and changes the efficiency for energy redistribution in the protein and the processes that drive nuclear spin relaxation. We also show that the Larmor frequency dependence of the protein-proton-spin-lattice relaxation rate constant is related to the frequency dependence of force constants and mean-square displacement commonly observed or calculated for proteins. We believe that these approaches give additional physical insight into the character of the extremely low-frequency protein dynamics.

Computer Simulation↗

PubMed2005

Nuclear magnetic resonance and spin relaxation in biological systems.

Proton nuclear spin-lattice relaxation in biological systems is generally distinguished from that in inorganic systems such as rocks by the presence of locally disordered macromolecular environments. Rapid exchange of readily observed labile small molecules among differently oriented macromolecular sites generally nearly averages the spectral anisotropies in the small molecule resonances. The biological tissue is generally distinguished from the inorganic matrix by the presence of a significant population of protons in the solid components that are well connected by dipolar spin couplings. Magnetic coupling between the solid and the liquid components generally dominates the magnetic field dependence of the spin-lattice relaxation rates observed in the small molecule components which is generally described by a power law in the Larmor frequency. Recent theory involving a modification of the spin-phonon class of relaxation mechanism provides a quantitative understanding of these data in terms of the dynamics of the chain molecules generally present in the solid spin systems, folded proteins for example.

Animals↗

PubMed2004

Local measures of intermolecular free energies in solution.

Proton spin-lattice relaxation rate changes induced by freely diffusing oxygen in aqueous and mixed solvents are reported for representative amino acids and glucose. The local oxygen concentration at each spectrally resolved proton was deduced from the paramagnetic contribution to the relaxation rate. The measured relaxation increment is compared to that of the force-free diffusion relaxation model, and the differences are related to a free energy for the oxygen association with different portions of the solute molecules. The free energy differences are small, on the order of -800 to -2000 J/mol, but are uniformly negative for all proton positions measured on the amino acids in water and reflect the energetic benefit of weak association of hydrophobic cosolutes. For glucose, CH proton positions report negative free energies for oxygen association, the magnitude of which depends on the solvent; however, the hydroxyl positions report positive free energy differences relative to the force-free diffusion model, which is consistent with partial occupancy in the OH region by a solvent hydrogen bond.

Amino Acids↗

PubMed2004

High frequency dynamics in hemoglobin measured by magnetic relaxation dispersion.

The magnetic relaxation dispersion profiles for formate, acetate, and water protons are reported for aqueous solutions of hemoglobin singly and doubly labeled with a nitroxide and mercury(II) ion at cysteines at beta-93. Using two spin labels, one nuclear and one electron spin, a long intramolecular vector is defined between the two beta-93 positions in the protein. The paramagnetic contributions to the observed 1H spin-lattice relaxation rate constant are isolated from the magnetic relaxation dispersion profiles obtained on a dual-magnet apparatus that provides spectral density functions characterizing fluctuations sensed by intermoment dipolar interactions in the time range from the tens of microseconds to approximately 1 ps. Both formate and acetate ions are found to bind specifically within 5 angstroms of the beta-93 spin-label position and the relaxation dispersion has inflection points corresponding to correlation times of 30 ps and 4 ns for both ions. The 4-ns motion is identified with exchange of the anions from the site, whereas the 30-ps correlation time is identified with relative motions of the spin label and the bound anion in the protein environment close to beta-93. The magnetic field dependence of the paramagnetic contributions in both cases is well described by a simple Lorentzian spectral density function; no peaks in the spectral density function are observed. Therefore, the high frequency motions of the protein monitored by the intramolecular vector defined by the electron and nuclear spin are well characterized by a stationary random function of time. Attempts to examine long vector fluctuations by employing electron spin and nuclear spin double-labeling techniques did not yield unambiguous characterization of the high frequency motions of the vector between beta-93 positions on different chains.

Acetates↗

PubMed2004

Magnetic relaxation dispersion of lithium ion in solutions of DNA.

The magnetic field dependence of the nuclear spin-lattice relaxation rate constant defines the magnetic relaxation dispersion (MRD) and provides a direct characterization of the molecular dynamics that cause fluctuations in the magnetic couplings in the system and may also indicate the dimensional constraints on the motion. The counterion cloud surrounding a linear polyelectrolyte ion, such as DNA in solution, provides an interesting opportunity for ion confinement that helps in understanding the thermodynamics and the dynamics of the interactions between the polyion and other solutes. The MRD profiles of lithium ion and tetramethylammonium ion were recorded in dilute aqueous solutions of native calf thymus DNA, which provides a long, charged rod that reorients slowly. The 7Li ion relaxes through the nuclear electric quadrupole coupling and the proton-lithium dipole-dipole coupling; the protons of the tetramethylammonium ion relax by dipole-dipole coupling. MRD profiles of the 7Li+ ion are dominated by transient interactions with the DNA that yield a linear dependence of the spin-lattice relaxation rate constant on the logarithm of the Larmor frequency. This magnetic field dependence is consistent with diffusive ion motions that modulate two spatial coordinates that characterize the relaxation couplings in the vicinity of the polyion. Motions around the rod and fluctuations in the ion distance from the rod are consistent with these constraints for lithium. The magnetic field dependence of the tetramethylammonium ion proton relaxation rate constant is weak, but also approximately a linear function of the logarithm of the Larmor frequency, which implies that the field dependence is caused in part by local order in the DNA solution.

Complex Mixtures↗

PubMed2004

Magnetic relaxation dispersion probe.

The magnetic field dependence of nuclear spin-lattice relaxation rates provides a powerful approach to characterizing intra and intermolecular dynamics. NMR spectrometers that provide extensive magnetic relaxation dispersion profiles may switch magnetic field strengths rapidly by either moving the sample or by changing the current in an electromagnet. If the sample is moved, the polarization and detection fields may be very high, which provides both high sensitivity and resolution. This report summarizes the design of a pneumatic sample transport system for glass sample containers that may be used in either a dual magnet spectrometer or in a single magnet system that exploits the fringe field as the secondary magnetic field.

Equipment Design↗

PubMed2004

Mapping oxygen accessibility to ribonuclease a using high-resolution NMR relaxation spectroscopy.

Paramagnetic contributions to nuclear magnetic spin-lattice relaxation rate constant induced by freely diffusing molecular oxygen measured at hundreds of different protein proton sites provide a direct means for characterizing the exploration of the protein by oxygen. This report focuses on regions of ribonuclease A where the rate constant enhancements are either quite large or quite small. We find that there are several regions of enhanced oxygen affinity for the protein both on the surface and in interior pockets where sufficient free volume permits. Oxygen has weak associative interactions with a number of surface crevices that are generally between secondary structural elements of the protein fold. Several regions near the surface have higher than expected accessibility to oxygen indicating that structural fluctuations in the protein provide intermolecular access. Oxygen penetrates part of the hydrophobic interior, but affinity does not correlate simply with hydrophobicity indices. Oxygen is excluded from regions of high interior packing density and a few surface sites where x-ray diffraction data have indicated the presence of specific hydration with high occupancy.

Binding Sites↗

PubMed2003

Protein reorientation and bound water molecules measured by 1H magnetic spin-lattice relaxation.

The water-proton spin-lattice relaxation rate constant, 1/T(1), was measured as a function of magnetic field strength for several dilute protein solutions. By separating the intermolecular contributions from the intramolecular contributions to the water-proton spin-lattice relaxation, the number of water molecules that bind to the protein for a time long compared with the rotational correlation time may be measured. We find a good correlation between the number of long-lived water molecules and the predictions based on available free volume in the proteins studied. The rotational correlation times of these proteins are larger than predicted by the Stokes-Einstein-Debye (SED) model for a sphere reorienting in a viscous liquid. The discrepancy between experiment and theory is usually attributed to hydration effects increasing the effective radius of the particle. However, the average lifetime of water molecules at the protein interface is far too short to justify such a picture. We suggest that surface roughness may be responsible for the retardation of rotational mobility and find that the SED model provides a reasonable representation of experiment if the radius assumed for the reorienting particle is the arithmetic mean of the crystallographic packing radius and the radius deduced from the effective surface area of the protein.

Animals↗

PubMed2003

End-to-end correlation for a C-12 hydrocarbon chain.

The 19F nuclear spin-lattice relaxation rate constants were measured as a function of magnetic field strength for 1,12-diaminododecane labeled at one end with a nitroxide radical and at the other with a trifluoromethyl group. The magnetic relaxation dispersion profile (MRD) reports the spectral density function appropriate to the end-to-end correlation function for the doubly labeled molecule. After extrapolation to zero concentration to eliminate the intermolecular relaxation contribution to relaxation, the resulting intramolecular MRD profile was compared with several model approaches. The rotational model for the spectral density functions as included in the Solomon-Bloembergen-Morgan equations does not describe the data well. The earlier model of Freed for nuclear spin relaxation induced by a freely diffusing paramagnetic co-solute is not rigorous for this case because the paramagnet is tethered to the observed nuclear spin and only a restricted space in the immediate vicinity of the nuclear spin is accessible for pseudo-translational diffusion of one end of the molecule with respect to the other. A generalization of the Torrey model for magnetic relaxation by translational diffusion developed by Nevzorov and Freed, which includes the effect of restrictions imposed by the finite length of the chain, describes the experiment within experimental errors. A simple modification of the Hwang-Freed model that does not specifically include the dynamical effects of the finite tether also provides a good approximation to the data when the tether chain is sufficiently long.

Carbon Isotopes↗

PubMed2002

O2 penetration and proton burial depth in proteins: applicability to fold family recognition.

Paramagnetically induced relaxation effects of O2 and the nitroxide 4-hydroxy TEMPO were measured for the amide protons of perdeuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium pasteurianum. For both O2 and the impermeant nitroxide, the induced relaxation at the static solvent inaccessible amide sites is dominated by long-range interactions with the paramagnetic species in the bulk aqueous phase. The upper bound of O2 solubility in the internal matrix of the rubredoxins is one-tenth that of the bulk aqueous phase. Furthermore, the difference between the oxygen solubilities inside the two rubredoxins is at most 1% that of bulk water O2 solubility, suggesting that there are only modest differences in this measure of fluidity for the mesophile vs hyperthermophile protein interiors. Calculations based on the assumption of a paramagnet uniformly distributed on the protein exterior yield accurate predictions at nearly all amide sites for the minimum relaxation value observed from either the O2 or nitroxide data. Model calculations indicate that the readily obtained paramagnetically induced relaxation effects should prove effective in recognition of structural homology for proteins that are too widely diverged for sequence-based recognition.

Bacterial Proteins↗

PubMed2002

Magnetic field dependence of proton spin-lattice relaxation times.

The magnetic field dependence of the water-proton spin-lattice relaxation rate (1/T(1)) in tissues results from magnetic coupling to the protons of the rotationally immobilized components of the tissue. As a consequence, the magnetic field dependence of the water-proton (1/T(1)) is a scaled report of the field dependence of the (1/T(1)) rate of the solid components of the tissue. The proton spin-lattice relaxation rate may be represented generally as a power law: 1/T(1)omega = A omega(-b), where b is usually found to be in the range of 0.5-0.8. We have shown that this power law may arise naturally from localized structural fluctuations along the backbone in biopolymers that modulate the proton dipole-dipole couplings. The protons in a protein form a spin communication network described by a fractal dimension that is less than the Euclidean dimension. The model proposed accounts quantitatively for the proton spin-lattice relaxation rates measured in immobilized protein systems at different water contents, and provides a fundamental basis for understanding the parametric dependence of proton spin-lattice relaxation rates in dynamically heterogeneous systems, such as tissues.

Magnetic Resonance Spectroscopy↗