Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate

In order to further understand the interaction processes between the Lycopodium alkaloids and acetylcholinesterase, the binding properties of N-acetyl huperzine A (1), huperzine B (2) and huperzine F (3) with Torpediniforms Nacline acetylcholinesterase (TnAchE) were investigated by 1H NMR methods. The nonselective, selective and double-selective spin-lattice relaxation rates were acquired in the absence and presence of TnAchE at a ratio of [ligand]/[protein]=1:0.005. The selective relaxation rates show protons of 1-3 have dipole-dipole interaction with protons of TnAchE at the binding interface. The molecular rotational correlation time of bound ligands was calculated by double-selective relaxation rate at 298 K, which showed that 1-3 had high affinity with the protein. The results indicate that investigation of 1H NMR relaxation data is a useful method to locate the new Lycopodium alkaloids as AchE inhibitors.     

NMR investigations of Mn(II)-leucine-enkephalin complexes in [2H6]-DMSO solution.

Structural and kinetic features of the Mn(II)-Leu-enkephalin binding equilibria were delineated by measuring 13C and 1H NMR spin-lattice relaxation rates. The temperature dependence of such rates showed that some carbons were experiencing slow exchange regimes such that kinetic parameters at room temperature could be calculated (k(off) = 1400 sec-1, delta H* = 12.0 kcal/mol, delta S* = -9.9 e.u.). The paramagnetic rates of fast exchanging carbons were interpreted by the Solomon-Bloembergen-Morgan theory to provide structural parameters. The terminal carboxyl and amino groups were shown to be the binding sites. The motional correlation time (tau c = 0.6 nsec at 298 K) was calculated by measuring selective and double-selective 1H spin-lattice relaxation rates for the free peptide. The number of coordinated ligands was evaluated by considering the distance of the Leu CO in the complex at 2.54 A, as shown by molecular models. Finally, carbon-Mn(II) distances were calculated and the molecular model of the 1:1 complex was built.     

Proton NMR T1, T2, and T1 rho relaxation studies of native and reconstituted sarcoplasmic reticulum and phospholipid vesicles.

The phospholipids protons of native and reconstituted sarcoplasmic reticulum (SR) membrane vesicles yield well-resolved nuclear magnetic resonance (NMR) spectra. Resonance area measurements, guided by the line shape theory of Bloom and co-workers, imply that we are observing a large fraction of the lipid intensity and that the protein does not appear to reduce the percent of the signal that is well resolved. We have measured the spin-lattice (T1) and spin-spin (T2) relaxation rates of the choline, methylene, and terminal methyl protons at 360 MHz and the spin-lattice relaxation rate in the rotating frame (T1 rho) at 100 MHz. Both the T1 and T2 relaxation rates are single exponential processes for all of the resonances if the residual water proton signal is thoroughly eliminated by selective saturation. The T1 and T2 relaxation rates increase as the protein concentration increases, and T2 rate decrease with increasing temperature. This implies that the protein is reducing both high frequency (e.g., trans-gauche methylene isomerizations) and low frequency (e.g., large amplitude, chain wagging) lipid motions, from the center of the bilayer to the surface. It is possible that spin diffusion contributes to the effect of protein on lipid T1's although some of the protein-induced T1 change is due to motional effects. The T2 relaxation times are observed to be near 1 ms for the membranes with highest protein concentration and approximately 10 ms for the lipids devoid of protein. This result, combined with the observation that the T2 rates are monophasic, suggests that at least two lipid environments exist in the presence of protein, and that the lipids are exchanging between these environments at a rate greater than 1/T2 or 10(3) s-1. The choline resonance yields single exponential T1 rho relaxation in the presence and absence of protein, whereas the other resonances measured exhibit biexponential relaxation. Protein significantly increases the single T1 rho relaxation rate of the choline peak while primarily increasing the T1 rho relaxation rate of the more slowly relaxing component of the methylene and methyl resonances.     

NMR relaxation of protein and water protons in diamagnetic hemoglobin solutions

We have measured T1 and T2 of protein and water protons in hemoglobin solutions using broad-line pulse techniques; selective excitation and detection methods enabled the intrinsic protein and water relaxation rates, as well as the spin-transfer rate between them, to be obtained at 5, 10, and 20 MHz. Water and protein T1 data were also obtained at 100 and 200 MHz for hemoglobin in H2O/D2O mixtures by using commercial Fourier-transform instruments. The T1 data conform to a simple model of two well-mixed spin systems with single intrinsic relaxation times and an average spin-transfer rate, with each phase recovering from a radio-frequency excitation with a biexponential time dependence. At low frequencies, protein T1 and T2 agree reasonably with a model of dipolar relaxation of an array of fixed protons tumbling in solution, explicitly calculating methyl and methylene relaxation and using a continuum approximation for the others. Differing values in H2O and D2O are mainly ascribed to solvent viscosity. For water-proton relaxation, T1, T2, and spin transfer were measured for H2O and HDO, which enabled a separation of inter-and intramolecular contributions to relaxation. Despite such detail, few firm conclusions could be reached about hydration water. But it seems clear that few long-lived hydration sites are needed to explain T1 and T2, and the spin-transfer value mandates fewer than five sites with a lifetime longer than 10(-8) s.     

A 300 MHz and 600 MHz proton NMR study of a 12 base pair restriction fragment: investigation of structure by relaxation measurements.

The 1H NMR spectrum of a 12 base pair DNA restriction fragment has been measured at 300 and 600 MHz and resonances from over 70 protons are individually resolved. Relaxation rate measurements have been carried out at 300 MHz and compared with the theoretical predictions obtained using an isotropic rigid rotor model with coordinates derived from a Dreiding model of DNA. The model gives results that are in excellent agreement with experiment for most protons when a 7 nsec rotational correlation time is used, although agreement is improved for certain base protons by using a shorter correlation time for the sugar group, or by increasing the sugar-base interproton distances. A comparison of non-selective and selective spin-lattice relaxation rates for carbon bound protons indicates that there is extensive spin diffusion even in this short DNA fragment. Examination of the spin-spin relaxation rates for the same type of proton on different base pairs reveals little sequence effect on conformation.     

Mapping the ligand binding site at protein side-chains in protein-ligand complexes through NOE difference spectroscopy

This report describes a novel NMR approach for mapping the interaction surface between an unlabeled ligand and a ¹³C,¹⁵N-labeled protein. The method relies on the spin inversion properties of the dipolar relaxation pathways and records the differential relaxation of two spin modes, where ligand and protein ¹H magnetizations are aligned either in a parallel or anti-parallel manner. Selective inversion of protein protons is achieved in a straightforward manner by exploiting the one-bond heteronuclear scalar couplings (¹J_CH, ¹J_NH). Suppression of indirect relaxation pathways mediated by bulk water or rapidly exchanging protons is achieved by selective inversion of the water signal in the middle of the NOESY mixing period. The method does not require deuteration of the protein or well separated spectral regions for the protein and the ligand, respectively. Additionally, in contrast to previous methods, the new experiment identifies side-chain enzyme ligand interactions along the intermolecular binding interface. The method is demonstrated with an application to the B₁₂-binding subunit of glutamate mutase from Clostridium tetanomorphum for which NMR chemical shift changes upon B₁₂-nucleotide loop binding and a high-resolution solution structure are available.     

The effect of cytochrome P-450cam on the NMR relaxation rate of water protons.

Cytochrome P-450cam in the native, substrate-free state (Fe3+, S = 1/2) substantially reduces the NMR relaxation times, T1 and T2, of water protons. Temperature and frequency dependences of T1 and T2 were measured; they are consistent with a model of one or two protons exchanging between a binding site on a heme ligand and bulk water. The relevant parameters of this model have been deduced from the data. The spin relaxation time of the heme iron, tau S similar to 0.5 ns at 25 degrees C, is unusually long for a low spin ferric heme protein but is compatible with the line widths measured for paramagnetically shifted heme resonances. The proton residence time on the ligand, tau M similar to 1 microsecond at 25 degrees C, follows an Arrhenius law with activation energy EM similar to 15 kcal/mol. A scalar hyperfine interaction A/h = 2.2 MHz (3.1 MHz for one-proton exchange) of the found proton(s) with the heme iron is deduced from the difference between T1 and T2 observed in the fast exchange limit. The iron-proton distance is found to be 2.9 A (2.6 A for one-proton exchange). Variation of pH between pH 6.4 and 8.6 does not affect T1. The bearing of these results on the question of the axial heme ligand is discussed.     

Nuclear magnetic resonance studies of substrate interaction with cobalt substituted alcohol dehydrogenase from liver.

The role of zinc in liver alcohol dehydrogenase has been studied by replacement of 1.3 and 3.5 of the four Zn(II) ions with Co(II) and measuring the effects of the paramagnetic Co(II) on the relaxation rates of the protons of water, ethanol, and isobutyramide. Water relaxation studies at 8, 24, 100, and 220 MHz indicate two classes of bound Co(II). The similar to 2 readily replaced Co(II) ions retain one fast exchanging water proton in their inner coordination spheres, while the similar to 2 slowly exchanging Co(II) ions coordinate no detectable water protons, indicating that the former replaced Zn(II) at the "catalytic sites" and the latter replaced Zn(II) at the "structural sites" detected crystallographically. Ethanol, acetaldehyde, and isobutyramide bind with appropriate affinities to the Co(II) substituted alcohol dehydrogenases decreasing the number of fast exchanging protons at the catalytic Co(II) site by greater than or equal to 54 percent. Coenzyme binding causes smaller changes in the water relaxation rate which may be due to local conformation changes. The paramagnetic effects of Co(II) at the catalytic site on the relaxation rates of the methyl protons of isobutyramide at 100 and 220 MHz indicate that this analog binds at a site 9.1 A from the catalytic Co(II). This distance decreases to 6.9 A when NADH is bound, and a Co(II) to methyne proton distance of 6.6 A is determined indicating a conformation change leading to the formation of a second sphere enzyme-Co(II)-isobutyramide complex in which a hydroxyl or water ligand intervenes between the metal and the substrate analog. Similar behavior is observed in the enzyme-ethanol complexes. The paramagnetic effects of Co(II), at the catalytic site, on the relaxation rates of the protons of ethanol at 100 and 220 MHz, indicate that this substrate bind at a site 12-14 A distant from the catalytic Co(II) but that this distancedecreases to 6.3 A in the abortive enzyme-NADH-ethanol complex. The role of the catalytic Co(II) thus appears to be the activation of a hydroxyl or water ligand which polarizes the aldehyde carbonyl group by hydrogen bonding. The role of the structural Co(II), which is more distant from isobutyramide (9-11 A), may be that of a template for protein conformation changes. By combining the present distances with those from previous magnetic resonance studies on the liver enzyme, the arrangement of coenzyme, metal, and substrate at the active site in solution can be constructed. This arrangement is consistent with that of ADP-ribose and zinc in the crystalline complex of liver alcohol dehydrogenase as determined by X-ray crystallography (Branden et al., (1973), Proc. Natl. Acad. Sci. U.S.A.70, 2439).     

Water Exchange at the Active Site of Carbonic Anhydrase: A Synthesis of the OH- and H2O-Models

We have measured the paramagnetic contribution to the magnetic relaxation rate of solvent protons in highly purified, buffer- and salt-free solutions of Co(2+)-substituted human carbonic anhydrase B (HCAB), as a function of pH in the range 5.5-10 and as a function of magnetic field. We have also measured the optical absorption at 640 nm to characterize the enzyme. The relaxation rates vary with pH much as does the CO(2) hydration activity, increasing with increasing pH. We find that the relaxation rates at all intermediate values of pH can be described as linear combinations of the rates obtained at the extremes of pH used, indicating the existence of low- and high-pH forms of the enzyme with pH-dependent concentrations. The optical data can be similarly represented. The fraction of high-pH form present, determined from either the relaxation or optical data, has a pK(a) of approximately 7.6 when approximated by a single ionization. The data are very similar to that for HCAB in the presence of buffer, in contrast to the bovine enzyme for which the pK(a) is affected substantially by the presence of sulfate. Previous analysis of the high relaxation rates at high pH indicated rapid exchange of Co(2+)-liganded protons, possible only if these exchanging protons were conveyed by water molecules. On the other hand, the present demonstration of the existence of two forms of HCAB in highly purified solutions, coupled with other data, argues strongly for ionization of a water molecule ligand of the metal ion at the active site, with OH(-) as the solvent-donated ligand at high pH. We propose a mechanism of ligand exchange at high pH that reconciles these ostensibly conflicting requirements by invoking a pentacoordinate intermediate having both OH(-) and H(2)O as ligands. Proton exchange can be rapid between these ligands because charge transfer without net ionization can occur, so that the leaving water can carry away the initial OH(-). The low-pH form is a thermal mixture of tetra- and pentacoordinate species, the latter having low relaxation rates by analogy with inhibitor derivatives of the enzyme and model systems. The proposed associative ligand-exchange mechanism reconciles the distinctions between the OH- and H(2)O-models of carbonic anhydrase by merging them, providing the first model is consistent with the observed pH dependence of hydration activity, optical absorption, and solvent magnetic relaxation.     

NMR studies of cytochrome P-450scc. Effects of steroid binding on water proton access to the active site of the ferric enzyme

Water proton relaxation rates of various complexes of cholesterol side chain cleavage cytochrome P-450 (-450scc) were investigated to gain information about the structure and dynamics of the steroid binding site. In all cases bulk water protons were found to be in rapid exchange with protons near the paramagnetic Fe3+ center, and the long electron spin relaxation time of the heme iron, tau s approximately 0.3 ns, resulted in fast relaxation rates. For the steroid-free enzyme, the closest approach of exchangeable protons is approximately 2.5 A, a distance consistent with a water molecule binding directly to the heme iron or rapidly exchanging with a coordinated ligand. When cholesterol was bound, the distance increased to approximately 4 A, indicative of displacement of water from the immediate coordination sphere of the heme but still in close proximity to the active site. For the complex with (22R)-22-hydroxycholesterol, a distance of approximately 2.7 A is observed, suggesting a reorganization of the active site when this intermediate is formed from cholesterol. Complexes of P-450scc with the competitive inhibitors (22R)-22-aminocholesterol, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, or (20R)-20-phenyl-5-pregnene-3 beta,20-diol, also yielded distances of approximately 2.5 A and reveal no effect of side chain size on access of protons to the heme. In the nitrogen-coordinated amino-steroid complexes, the distances observed indicate solvent proton exchange with the heme-bound nitrogen ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies on the copper(II) complexes of carnosine

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Measurement of amide hydrogen exchange rates with the use of radiation damping

A simple method for measuring amide hydrogen exchange rates is presented, which is based on the selective inversion of water magnetization with the use of radiation damping. Simulations show that accurate exchange rates can be measured despite the complications of radiation damping and cross relaxation to the exchange process between amide and water protons. This method cannot eliminate the contributions of the exchange-relayed NOE and direct NOE to the measured exchange rates, but minimize the direct NOE contribution. In addition, the amides with a significant amount of such indirect contributions are possible to be identified from the shape of the exchange peak intensity profiles or/and from the apparent relaxation rates of amide protons which are extracted from fitting the intensity profiles to an equation established here for our experiment. The method was tested on ubiquitin and also applied to an acyl carrier protein. The amide exchange rates for the acyl carrier protein at two pHs indicate that the entire protein is highly dynamic on the second timescale. Low protection factors for the residues in the regular secondary structural elements also suggest the presence of invisible unfolded species. The highly dynamic nature of the acyl carrier protein may be crucial for its interactions with its substrate and enzymes.     

Protein global fold determination using site-directed spin and isotope labeling

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 A were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 A from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.     

Dynamics of erabutoxin b as studied by nuclear magnetic resonance. Relaxation studies of methyl proton resonances

Longitudinal and transverse relaxation times were measured for well-resolved and assigned methyl proton resonances of erabutoxin b at 270 MHz, 300 MHz and 500 MHz. Both longitudinal and transverse magnetization decay curves are non-exponential due to cross-relaxation and cross-correlation effects. The longitudinal and transverse relaxation rates were obtained from the initial slope of both magnetization decay curves. The correlation times for the isotropic tumbling motion of the protein were determined to be 2.82 ns at 300 K and 1.62 ns at 330 K from the analysis of the relaxation data of some alpha protons. Using these values, the relaxation data of methyl protons were fitted to various theoretical models. Most of the methyl resonances could be fitted well to a model which allowed methyl rotation (in the range 0.01-0.05 ns) and an external contribution from protons assumed to be in positions derived from X-ray coordinates. The data for a few methyl groups, however, could not be fitted in this way. For these a smaller number of external protons than predicted by the X-ray coordinates was assumed. Additionally, a larger amplitude motion had to be introduced into the model for particular residues. This additional motion requires concerted protein motion close to these residues, since the X-ray structure suggests that steric hindrance would prevent local motion. These results are consistent with the idea of a flexible and dynamic structure for proteins.     

Copper(II) complexes of carnosine, glycylglycine, and glycylglycine-imidazole mixtures

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

Proton and deuteron nuclear magnetic relaxation dispersion studies of Ca2+-Mn2+-concanavalin A: evidence for two classes of exchanging water molecules

We have measured the magnetic field dependence of the nuclear magnetic relaxation rates (NMRD profiles) of solvent protons and deuterons in solutions of Ca2+-Mn2+-concanavalin A (Con A) with and without saccharide present. Data were obtained over the range -8 to 35 degrees C; the extension to the lowest temperature was made possible by the presence of 5 M salt. Since previous theoretical analyses, using accepted relaxation theories of 1H NMRD profiles alone, led to unsatisfactory conclusions, we have attempted to take advantage of the fact that the residence lifetime of a water ligand of the metal ions can influence the relaxation behavior of protons and deuterons differently. From a comparison of the present proton and deuteron results, we find that Ca2+-Mn2+-Con A has two classes of binding sites: one, associated with the inner coordiation sphere of the Mn2+ ions, having a resident lifetime for solvent water of approximately 10(-5) s that is reduced by the presence of saccharide and another having a lifetime of approximately 5 X 10(-9) s, located with the protons of the bound waters approximately 4.4 A from the Mn2+ ions (assuming two equivalent water molecules in this class), which is well beyond the coordination environment of the Mn2+ ions. The relaxation contribution of these more distant sites is unaffected by saccharide. The conclusions are corroborated by measurements of the temperature dependences of the proton NMRD profiles, which show quite clearly that the profiles are composite, containing two contributions with opposite dependences on temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of the backbone protons and secondary structure of pentapeptide and heptapeptide substrates bound to bovine heart protein kinase

The conformations of enzyme-bound pentapeptide (Arg-Arg-Ala-Ser-Leu) and heptapeptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) substrates of protein kinase have been studied by NMR in quaternary complexes of the type (Formula: see text). Paramagnetic effects of Mn2+ bound at the inhibitory site of the catalytic subunit on the longitudinal relaxation rates of backbone Ca protons, as well as on side-chain protons of the bound pentapeptide and heptapeptide substrates, have been used to determine Mn2+ to proton distances which range from 8.2 to 12.4 A. A combination of the paramagnetic probe-T1 method with the Redfield 2-1-4-1-2 pulse sequence for suppression of the water signal has been used to measure distances from Mn2+ to all of the backbone amide (NH) protons of the bound pentapeptide and heptapeptide substrates, which range from 6.8 to 11.1 A. Paramagnetic effects on the transverse relaxation rates yield rate constants for peptide exchange, indicating that the complexes studied by NMR dissociate rapidly enough to participate in catalysis. Model-building studies based on the Mn2+-proton distances, as well as on previously determined distances from Cr3+-AMPPCP to side-chain protons [Granot, J., Mildvan, A.S., Bramson, H. N., & Kaiser, E. T. (1981) Biochemistry 20, 602], rule out alpha-helical, beta-sheet, beta-bulge, and all possible beta-turn conformations within the bound pentapeptide and heptapeptide substrates. The distances are fit only by extended coil conformations for the bound peptide substrates with a minor difference between the pentapeptides and heptapeptides in the phi torsional angle at Arg3C alpha and in psi at Arg2C alpha. An extended coil conformation, which minimizes the number of interactions within the substrate, would facilitate enzyme-substrate interaction and could thereby contribute to the specificity of protein kinase.     

Nmr study of collagen-water interactions

A proton magnetic resonance study of different cross-linked collagens was performed as a function of water content and temperature. Collagens from three connective tissues (calf, steer, and cow) were chosen according to the different number of nonreducible multivalent cross-links, which increases during the life of animal. Samples were hydrated under five well-defined water activities (A_w) ranging from 0.44 to 0.85. The transverse and cross-relaxation times of water protons were studied as a function of temperature from ?20 up to 100°C. From the temperature dependence of relaxation rates, the dynamics of water molecules can be described according to different processes: exchange of protons at the higher temperatures and dipole-dipole interactions that prevail at the lower temperatures. The exchange processes are analyzed as a function of the residence lifetime of water molecules at the protein interface and of the transfer of spin energy from water protons to macromolecule protons. The proton dipole-dipole interactions are related to the relaxation parameters of protein and water protons. All the relaxation parameters showed specific behavior for the 0.44 water activity for every tissue. The collagen tissue from calf also showed distinct behavior in comparison with other tissues. © 1994 John Wiley & Sons, Inc.     

Magnetic cross-relaxation among protons in protein solutions.

The magnetic spin-lattice relaxation rates of solvent water nuclei are known to increase upon addition of diamagnetic solute protein. This enhancement of the relaxation rate is a function of magnetic field, and the orientational relaxation time of the protein molecules can be deduced from analysis of the field-dependent relaxation rates. Although the nature of the interactions that convey information about the dynamics of protein motion to the solvent molecules is not established, it is known that there is a contribution to the relaxation rates of solvent protons that plays no role in the relaxation of solvent deuterons and 17O nuclei. We show here that the additional interaction arises from a cross-relaxation process between solvent and solute protons. We introduce a heuristic three-parameter model in which protein protons and solvent protons are considered as two separate thermodynamic systems that interact across the protein-solvent interface. The three parameters are the intrinsic relaxation rates of each system and a cross-relaxation term. The sign of the latter term must always be positive, for all values of magnetic field, in order for magnetization energy to flow from the hotter to the cooler system. We find that the magnetic field-dependence of the cross-relaxation contribution is much like that of the remaining solvent proton relaxation, i.e., about the same as the deuteron relaxation field dependence. This finding is not compatible with the predictions of expressions for the cross-relaxation that have been used by other authors, but not applied to data over a wide range of magnetic field strength. The model predicts that the relaxation behavior of both the protein protons and the solvent protons is the sum of two exponentials, the relative contributions of which would vary with protein concentration and solvent isotopic composition in a fashion suggestive of the presence of two classes of protein protons, when there is in reality only one. This finding has immediate implications for the interpretation of published proton relaxation rates in complex systems such as tissues; these data should be reexamined with cross-relaxation taken into account.     

1H and 31P Fourier transform magnetic resonance studies of the conformation of enzyme-bound propionyl coenzyme A on the transcarboxylase.

The relaxation rates of the carbon-bound protons and of the three assigned phosphorus resonances of propionyl-CoA were measured in solutions of free propionyl-CoA and of the transcarboxylase-propionyl-CoA complex. In free propionyl-CoA, analysis of the 1/T1 values of 15 protons at 100 and 220 MHz and of 1/T1 and 1/T2 of the three phosphorus atoms at 40.5 MHz indicated free rotation of the propionyl region (taur approximately 3 x 10(-11) sec) but hindered motion of the remainder of the molecule with correlation times of 1-3. 5 x 10(-10) sec, approaching the tumbling time of the entire molecule (taur - 6 x 10(-10) sec. The correlation times of the three phosphorus atoms were indistinguishable from those of their nearest neighbor protons. The effects of three homogeneous enzyme preparations with varying contents of Zn(II), Co(II), and Cu(II) on 1/T1 of 12 protons and 3 phosphorus atoms of prionyl-CoA were analyzed with the help of simultaneous equations to yield the individual contributions at the three metal sites. Only diamagnetic effects were detected on the relaxation rates of the three phosphorus atoms. From the diamagnetic effects it was calculated that the motions of the prionyl side chain and of the terminal pantetheine methylene protons were hindered on the enzyme by an order of magnitude (taur approximately 6 x 10(-10) sec) and that the phosphorus atoms were hindered by two orders of magnitude (taur approximately 1 x 10(-8) sec) over the taur values found in free propionyl-CoA, but that these taur values remained well below that of the entire protein molecule (taur =6 x 10(-7) sec)...