Magnetic resonance distinction between site bound and atmospherically bound paramagnetic counterions in polyelectrolyte solutions.

A combination of the water protons NMR chemical shifts, longitudinal and transversal relaxation rates and of the paramagnetic counterion EPR signal is shown to provide a clear distinction between site binding, atmospheric trapping and free counterions in solutions of polyelectrolyte TMA salts with increasing concentrations of the divalent counterions Co++ and Mn++. Site binding is defined by the loss of water in the counterion first hydration shell while atmospheric binding results in a change in the counterion correlation time as compared to a free ion.     

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)     

Ultrasonic study of the kinetics of counterion site binding in aqueous solutions of polyelectrolytes

The excess ultrasonic absorption due to counterion binding has been studied as a function of frequency for a series of polysalts in the range 1–150 MHz. All the relaxation spectra can be represented by a relaxation equation with two relaxation terms. The relaxation frequencies appear concentration independent and the relaxation amplitudes seem proportional to concentration. The low frequency relaxation process appears to depend mainly on the nature of the counterion while the high frequency relaxation process seems to be mostly dependent on the nature of the polyion. These results are quite similar to those obtained in ultrasonic studies of ion-pairing in solutions of divalent sulfates. The kinetic model used for the quantitative analysis of these results has been modified for polysalts through introducing the concept of“counterion condensation”. In this modified model the excess absorption is assigned to the perturbation by the ultrasonic waves of the equilibria between the three states of hydration of ths complex formed by a counterion and that part of the polyion where it is bound. Analytical expressions of the relaxation amplitudes have been derived using classical procedures for this modified kinetic model. In the case of cobalt-polyphosphate (Co-PP), the ultrasonic data together with the results of NMR measurements on either Co²⁺ or Co-PP have been used for the evaluation of the volume changes, the rate constants and the fractions of counterions in the three states of hydration involved in the binding equilibria. The volume changes obtained in this manner depend only slightly on the method of calculation and appear to be consistent with volume changes for outer-sphere and inner-sphere complex formation. These results are discussed.     

Solution NMR of water on protein surfaces

Specialized water-suppression NMR pulse sequences and heteronuclear three-dimensional NMR have now made it possible to observe cross relaxation between the protons of water molecules and protons on the surface or in the interior of biological molecules. The cross-relaxation characteristics at each site allow loosely and tightly bound waters to be distinguised and limits on the residence lifetimes to be placed. These observations are affording a new view of the nature of protein hydration.     

Water binding and mobility in the phosphatidylcholine/cholesterol/water lamellar phase

Measurements of hydration and water self diffusion in lamellar phases of the ternary system: phosphatidylcholine/cholesterol/water have been made using pulse NMR relaxation methods. Systems containing phosphatidylcholine and cholesterol in a 1 : 1 mol ratio with varying water contents are studied at 20.5°C. The results indicate that 12 water molecules corresponds to complete hydration of the phosphatidylcholine/cholesterol unit, and in the region of this hydration a 4-fold decrease in water diffusion occurs. The nature of the bound water and its relationship to phase stability and overall water mobility in the system are discussed. It is concluded that at the stoichiometric composition the diffusion decreases due to the relative immobility of the bound water. The implications in terms of permeability regulation in the aqueous channels by water content and hydration are cited.     

Water binding and mobility in the phosphatidylchlone/cholesterol/water lamellar phase.

Measurements of hydration and water self diffusion in lamellar phases of the ternary system: phosphatidylcholine/cholesterol/water have been made using pulse NMR relaxation methods. Systems containing phosphatidylcholine and cholesterol in a 1:1 mol ratio with varying water contents are studied at 20.5 degrees C. The results indicate that 12 water molecules corresponds to complete hydration of the phosphatidylcholine/cholesterol unit, and in the region of this hydration a 4-fold decrease in water diffusion occurs. The nature of the bound water and its relationship to phase stability and overall water mobility in the system are discussed. It is concluded that at the stoichiometric composition the diffusion decreases due to the relative immobility of the bound water. The implications in terms of permeability regulation in the aqueous channels by water content and hydration are cited.     

Comparative study of glutamine synthetase bound lanthanide(III) ions using NMR relaxation and lanthanide(III) luminescence techniques

Changes in the intrinsic fluorescence intensity of glutamine synthetase induced by lanthanide(III) ion binding demonstrate the existence of three types of sites for these ions. The sites are populated sequentially during titrations of the enzyme, and the first two have a stoichiometry of 1 per enzyme subunit. The number of water molecules coordinated to Eu(III) bound to the first site was determined by luminescence lifetime techniques to be 4.1 +/- 0.5. The hydration of Gd(III) bound to the same site was studied by magnetic field dependent water proton longitudinal relaxation rate measurements, and by water proton and deuteron relaxation measurements of one sample at single magnetic fields. The magnetic resonance techniques also yield a value of 4 for the hydration number.     

Rotational and translational water diffusion in the hemoglobin hydration shell: dielectric and proton nuclear relaxation measurements.

The dynamic properties of water in the hydration shell of hemoglobin have been studied by means of dielectric permittivity measurements and nuclear magnetic resonance spectroscopy. The temperature behavior of the complex permittivity of hemoglobin solutions has been measured at 3.02, 3.98, 8.59, and 10.80 GHz. At a temperature of 298 K the average rotational correlation time tau of water within a hydration shell of 0.5-nm thickness is determined from the activation parameters to be 68 +/- 10 ps, which is 8-fold the corresponding value of bulk water. Solvent proton magnetic relaxation induced by electron-nuclear dipole interaction between hemoglobin bound nitroxide spin labels and water protons is used to determine the translational diffusion coefficient D(T) of the hydration water. The temperature dependent relaxation behavior for Lamor frequencies between 3 and 90 MHz yields an average value D(298K) = (5 +/- 2) x 10(-10)m2 s-1, which is about one-fifth of the corresponding value of bulk water. The decrease of the water mobility in the hydration shell compared to the bulk is mainly due to an enhanced activation enthalpy.     

1H nuclear magnetic resonance studies of the conformation of an ATP analogue at the active site of Na,K-ATPase from kidney medulla

1H nuclear magnetic relaxation measurements have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase [Klevickis, C., & Grisham, C. M. (1982) Biochemistry 21, 6979; Gantzer, M. L., Klevickis, C., & Grisham, C. M. (1982) Biochemistry 21, 4083] and that Mn2+ bound to a single, high-affinity site on the ATPase can be an effective paramagnetic probe for nuclear relaxation studies of the Na,K-ATPase [O'Connor, S. E., & Grisham, C. M. (1979) Biochemistry 18, 2315]. From the paramagnetic effect of Mn2+ bound to the ATPase on the longitudinal relaxation rates of the protons of Co(NH3)4ATP at the substrate site (at 300 and 361 MHz), Mn-H distances to seven protons on the bound nucleotide were determined. Taken together with previous 31P nuclear relaxation data, these measurements are consistent with a single nucleotide conformation at the active site. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. The glycosidic torsion angle is 35 degrees, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The bound Mn2+ lies above and in the plane of the adenine ring. The distances from Mn2+ to N6 and N7 are too large for first coordination sphere complexes but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR spin grouping and correlation exchange analysis. Application to low hydration NaDNA paracrystals.

The NMR spin-grouping technique is applied to low hydration oriented fibers of NaDNA to study the role of exchange in determining the apparent (observed) spin relaxation of the system. The analysis proceeds in three steps: first, the apparent proton relaxation is measured at high fields, with both selective and nonselective inversion pulse sequences, and in the rotating frame. The spin-grouping technique is used in all spin-lattice relaxation measurements to provide the optimum apparent relaxation characterization of the sample. Next, all apparent results are analyzed for exchange. In this analysis the results from the high field and rotating frame experiments (which probe the exchange at two different time scales) are correlated to determine the inherent (or true) spin relaxation parameters of each of the proton groups in the system. The results of selective inversion T1 measurements are also incorporated into the exchange analysis. Finally, the dynamics of each spin group are inferred from the inherent relaxation characterization. The low hydration NaDNA structure is such that the exchange between the protons on the water and those on the NaDNA is limited, a priori, to dipolar mixing. The results of the exchange analysis indicate that the dipolar mixing between water and NaDNA protons is faster than the spin diffusion within the NaDNA proton group itself. The spin-diffusion on the macromolecule is the bottleneck for the exchange between the water protons and the NaDNA protons. The water protons serve as the relaxation sink both at high fields and in the rotating frame for the total NaDNA-water spin bath. The inherent relaxation of the water is characteristic of water undergoing anisotropic motion with a fast reorientational correlation time about one axis (5 X 10(-10) less than or equal to tau r less than or equal to 8 X 10(-9)S) which is about three orders of magnitude slower than that of water in the bulk; and a slow tumbling correlation time for this axis (1.5 x 10(-7) less than or equal to tau t less than or equal to 8 x 10(-7)S) which is two orders of magnitude slower yet.     

Molecular dynamics investigation of the interaction between DNA and distamycin

The complex of the minor groove binding drug distamycin and the B-DNA oligomer d-(CGCAAATTTGCG) was investigated by molecular dynamics simulations. For this purpose, accurate atomic partial charges of distamycin were determined by extended quantum chemical calculations. The complex was simulated without water but with hydrated counterions. The oligomer without the drug was simulated in the same fashion and also with 1713 water molecules and sodium counterions. The simulations revealed that the binding of distamycin in the minor groove induces a stiffening of the DNA helix. The drug also prevents a transition from B-DNA to A-DNA that was found to occur rapidly (30 ps) in the segment without bound distamycin in a water-free environment but not in simulations including water. In other simulations, we investigated the relaxation processes after distamycin was moved from its preferred binding site, either radially or along the minor groove. Binding in the major groove was simulated as well and resulted in a bound configuration with the guanidinium end of distamycin close to two phosphate groups. We suggest that, in an aqueous environment, tight hydration shells covering the DNA backbone prevent such an arrangement and thus lead to distamycin's propensity for minor groove binding.     

Arrangement of the substrates at the active site of brain pyridoxal kinase

The distances between enzyme-bound paramagnetic CrATP (a stable, beta, gamma-bidentate complex of Cr3+ and ATP) at the active site of sheep brain pyridoxal kinase and the protons of bound inhibitor 4-dPyr (4-deoxypyridoxine) were determined in the ternary enzyme-CrATP.4-dPyr complex by measuring the paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T1p) of the protons of 4-dPyr. The correlation time for the Cr(3+)-4-dPyr dipolar interaction on the enzyme was estimated as 1.59 ns by the frequency dependence of 1/T1p of water protons. Temperature dependence of 1/T1p values indicated the fast exchange of 4-dPyr from the paramagnetic enzyme.CrATP.4-dPyr complex; hence the measured 1/T1p values can be used for metalnucleus distance determinations. The distances from the Cr3+ of the enzyme-bound CrATP to the 2-methyl (7.19 A), 4-methyl (7.18 A), and H6 proton (6.18 A) of the 4-dPyr are too great to permit a direct coordination of any group from 4-dPyr. However, these distances can be built into a model in which phosphorus of the gamma-phosphoryl group of ATP is 4 A away from the oxygen atom of the 5-CH2OH group of the 4-dPyr. This suggests that phosphorylation of pyridoxal can occur via direct transfer of the phosphoryl group between the bound substrates at the active site of pyridoxal kinase.     

Time-domain reflectrometry studies of water binding and structural flexibility in chymotrypsin

Time-domain dielectric spectroscopy has been employed to probe the hydration properties and structural flexibility of chymotrypsin (EC 3.4.21.1). The dielectric properties of the hydrated protein above 100 MHz have been used to identify two categories of protein-bound water, the first being irrotationally bound to the protein with a second, relatively weakly bound, having a rotational freedom comparable with that of normal bulk water. A dielectric dispersion observed, centred at 12 MHz, has been attributed to the relaxation of the polar components of the protein structure. This dielectric loss became increasingly significant above a transition in the hydration dependence, where water is relatively weakly bound to the chymotrypsin. This is discussed in terms of the formation of water clusters on the protein surface which screen electrostatic interactions between protein-charged groups.     

Protein-bound water molecule counting by resolution of (1)H spin-lattice relaxation mechanisms

Water proton spin-lattice relaxation is studied in dilute solutions of bovine serum albumin as a function of magnetic field strength, oxygen concentration, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurately Lorentzian with an effective correlation time of 41 +/- 3 ns when measured at low proton and low protein concentrations to minimize protein aggregation. Elimination of oxygen flattens the relaxation dispersion profile above the rotational inflection frequency, nearly eliminating the high field tail previously attributed to a distribution of exchange times for either whole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a time scale of order one ns. Measurements as a function of isotope composition permit separation of intramolecular and intermolecular relaxation contributions. The magnitude of the intramolecular proton-proton relaxation rate constant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correlation time of 42 ns. This number of bound water molecules neglects the possibility of local motions of the water in the binding site; inclusion of these effects may increase the number of bound water molecules by 50%.     

Water in agarose gels studied by nuclear magnetic resonance relaxation in the rotating frame.

The dependence of the spin-lattice relaxation time in the rotating frame (T1rho) on radio frequency (RF) field strength and temperature has been studied for agarose gels in order to investigate molecular motion. The results indicate the presence of slow motions with a correlation time of ca. 5-10(-6) s at room temperature. This interaction is responsible for the short spin-spin relaxation times (T2) for water protons in agarose gels and is ascribed to firmly bound water. The fraction of bound water is estimated to about 0.003 for a 7.3% agarose gel. The motion of the more mobile protons in agarose-water systems can not be characterized by single correlation time. This fraction is presumably composed of water in different motional states and some of the agarose hydroxyl protons. Higher mobilities are the most common.     

A novel nuclear relaxation approach for estimating distance between enzyme- and nucleotide-bound metal ions at the catalytic site of pyruvate kinase.

This communication introduces a nuclear relaxation approach for an estimation of the distance between two paramagnetic metal ion sites on a metal-activated enzyme. The method is based on the existence of an exchange of unpaired electron spin magnetizations between the two metals via energy-conserving concerted mutual spin flips which arise from time-dependent dipolar interactions of the electronic magnetizations. This cross-relaxation of electronic magnetizations depends on the inverse sixth power of the intermetal distance and may, under suitable conditions, affect the longitudinal relaxation rate of inner sphere water protons by altering the electron-proton dipolar correlation time when the latter is dominated by electron spin relaxation. The technique is applied to estimate the distance of 5.2 +/- 0.9 A between Mn2+ and Cr3+ in the pyruvate kinase-Mn2+-ATPCr3+ complex and indicates the existence of a van der Waals contact between the hydration spheres of the enzyme- and nucleotide-bound metal ions.     

A theoretical study oh the effect of "bound" water on the proton chemical shifts of the nucleic acid bases.

Computations are performed on the proton chemical shifts due to hydrogen bonding between the purine and pyrimidine bases of the nucleic acids and water molecules of their first hydration shell. The water molecules should produce measurable shifts essentially for protons of the bases located close to the site of interaction. For the imino protons of the bases G-N1H and U-N3H participating in hydrogen bonding, the calculated delta delta is larger for the interaction of a base with a complementary base than for its interaction with water. Base pairing will thus produce a downfield shift in water but the measured delta delta due to pairing in this solvent will be smaller than in an inert solvent. Also, the chemical shift difference between G-N1H and U-N3H in water will be larger if the molecules are engaged in pairs than if they are not.     

NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme

Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual consistency of interproton and Cr3+ to proton distances obtained in metal-ATP complexes of both the enzyme and the peptide suggests that the conformation of the peptide is very similar to that of residues 1-45 of the enzyme. When this was assumed to be the case and when molecular models and a computer graphics system were used, MgATP could be fit into the X-ray structure of adenylate kinase in a unique manner such that all of the distances determined by NMR were accommodated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aconitase: its source of catalytic protons

An ordinary isotope partition experiment was performed to determine the rate of dissociation of the proton from the donor site for the hydration of cis-aconitate. Aconitase in [3H]water was efficiently diluted into well-mixed solutions of cis-aconitate. Citrate and isocitrate that were formed within 2 s were more heavily labeled than could be explained by consideration of an isotope effect in the processing of one proton per enzyme equivalent. Control experiments indicate that mixing was much more rapid than catalytic turnover, ruling out incompletely diluted [3H]water as a significant isotope source. Therefore, it appears that significantly more than one enzyme-bound tritium atom (protons) must have been used in the course of the multiple turnover of the enzyme after the dilution was complete. Isotope incorporation reached values in excess of four proton equivalents as a limit with simple Michaelis dependence on cis-aconitate. From the half-saturation concentration value for trapping, 0.15 mM, the t 1/2 for exchange of each of these protons with solvent appears to be approximately 0.1 s at 0 degrees C. The large number of protons trapped seems to suggest the existence of a structurally stabilized pool of protons, or water, that communicates between the active site base and the medium in the hydration of cis-aconitate. The proton abstracted in the dehydration of [3H]citrate is transferred directly to undissociated cis-aconitate to form isocitrate without dilution, or cis-aconitate having dissociated, the tritium passes to the medium, presumably through the pool of bound protons indicated above. All of the citrate-derived protons can be found in isocitrate if cis-aconitate is added in sufficient concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnetic resonance studies of the proximity and spatial arrangement of propionyl coenzyme A and pyruvate on a biotin-metalloenzyme, transcarboxylase.

A spin-labeled ester of CoA, R-CoA (3-carboxy-2,2,5,5-tetramethyl-1-pyrolidinyl-1-oxy CoA thioester), has been shown by competition studies using electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) to bind specifically to the propionyl-CoA binding sites of transcarboxylase. Titrations indicate 0.7 +/- 0.2 binding site for R-CoA per enzyme-bound biotin with a dissociation constant of 0.33 +/- 0.12 mM. Propionyl-CoA binds to this site with a 1.3-fold lower disonable agreement with kinetically determined inhibitor constants of CoA and propionyl-CoA and propionyl-CoA (D. B. Northrop (1969), J. Biol. Chem. 244, 5808). The bit of this spin-label on 1/T1 of water protons. The formation of a ternary transcarboxylase-R-CoA-pyruvate complex is suggested by the failure of pyruvate to displace R-CoA from the tight site and is established by the paramagnetic effects of enzyme-bound R-CoA on the relaxation rates of the protons and 13C atoms of enzyme-bound pyruvate. From the paramagnetic effects of R-CoA on the relaxation rates of the methyl protons of pyruvate at 40.5 and 100 MHz, and on the 13C-enriched carbonyl and carboxyl carbon atoms of pyruvate at 25.1 MHz, a correlation time of 7 nsec and distances from the bound nitroxide radical to the methyl protons, the carbonyl, and carboxyl carbon atoms of bound pyruvate of 7.9 +/- 0.7, 10.3 +/- 0.8, and 12.1 +/- 0.9 A, respectively, are calculated. These distances establish the close proximity of the CoA ester and keto acid sites on transcarboxylase. Together with the previously determined distances from the enzyme-bound (Co(II) to the methyl protons and 2 carbon atoms of bound pyruvate and to 12 protons and 3 phosphorus atoms of bound propionyl-CoA, the present distances are used to derive a composite model of the bound substrates in the overall transcarboxylation reaction. In this model the distance from the methyl carbon of pyruvate and the methylene carbon of propionyl-CoA, between which the carboxyl transfer takes place is only approximately 7 A. Depending on the detailed mechanism of the carboxyl transfer, the distance through which the carboxybiotin must migrate is therefore between 0 and 7 A. Hence the major role of the 14-A arm of carboxybiotin is not to permit a large carboxyl migration but, rather to permit carboxybiotin to traverse the gap which occurs at the interface of three subunits and to insinuate itself between the CoA and keto acid sites.