Association of ferri- and ferro-cytochrome c with lipid multilayers: a 31P solid-state NMR study

The 31P nuclear magnetic resonance anisotropies of dispersions of diacylphosphatidic acid and diacylphosphatidylserine were slightly increased in the presence of cytochrome c: no increase was observed with cardiolipin. However, the 31P spin-lattice relaxation times (T1) for all of these lipids were reduced markedly by the protein. As similar effects were observed with ferri-cytochrome c and with the reduced protein, which is diamagnetic, we suggest that the changes in T1 reflect a reduction in the spectral density of fast motions for the lipid headgroups attendant on binding of protein, rather than paramagnetic relaxation of the phosphorus nuclear spin.     

Molecular flexibility demonstrated by paramagnetic enhancements of nuclear relaxation. Application to alamethicin: a voltage-gated peptide channel.

A nitroxide spin label attached to the C-terminus of the channel forming peptide alamethicin produces an enhancement of the nuclear spin-lattice relaxation rates of peptide protons as a result of both intermolecular and intramolecular magnetic dipole-dipole interactions. The intermolecular contribution provides evidence that alamethicin monomers collide preferentially in a C-terminal-to-N-terminal configuration in methanol. From the intramolecular paramagnetic enhancement of nuclear spin-lattice relaxation times, effective distances between the unpaired electron on the nitroxide at the C-terminus of alamethicin and protons along the peptide backbone were calculated. These distances are much shorter than distances based on the reported crystal structure of alamethicin, and cannot be accounted for by motion in the bonds that attach the nitroxide to the peptide. In addition, the differences between distances deduced from the nuclear spin relaxation and the distances seen in the crystal structure increase toward the N-terminal end of the peptide. The simplest explanation for these data is that the alamethicin backbone suffers large structural fluctuations that yield shorter effective distances between the C-terminus and positions along the backbone. This finding can be interpreted in terms of a molecular mechanism for the voltage-gating of the alamethicin channel. When the distances between a paramagnetic center and a nucleus fluctuate, paramagnetic enhancements are expected to yield distances that are weighted by r-6, and distances calculated using the Solomon-Bloembergen equations may more nearly represent a distance of closest approach than a time average distance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes.

The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions.     

Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.     

Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.     

The distribution of lipid attached spin probes in bilayers: application to membrane protein topology

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.     

Nuclear magnetic relaxation dispersion study of the dynamics in solid homopolypeptides

The (1)H nuclear magnetic relaxation dispersion profiles were measured from 10 kHz to 30 MHz as a function of temperature for polyglycine, polyalanine, polyvaline, and polyphenylalanine to examine the contributions of different side chain motions to the polypeptide proton relaxation rate constants. The spin-fracton theory for (1)H relaxation is modified to account for high frequency motions of side chains that are dynamically connected to the linear polymer backbone. The (1)H relaxation is dominated by propagation of rare disturbances along the backbone of the polymer. The side-chain dynamics cause an off-set in the field dependence of the (1)H spin-lattice relaxation rate constants which obey a power law in the Larmor frequency in the limit of low and high magnetic field strength.     

Nuclear magnetic spin-lattice relaxation of water protons caused by metal cage compounds.

The nuclear magnetic spin-lattice relaxation rates of water protons are reported for solutions of manganese(II), copper(II), and chromium(III) cage complexes of the sarcophagine type. As simple aqueous solutions, the complexes are only modest magnetic relaxation agents, presumably because they lack protons on atoms in the first-coordination-sphere protons that are sufficiently labile to mix the large relaxation rate at the metal complex with that of the bulk solvent. The relaxation is approximately modeled using spectral density functions derived for translational diffusion of the interacting dipole moments with the modification that the electron spin relaxation rate is directly included as a contribution to the correlation time. In all cases studied, the electron spin relaxation rate is sufficiently large that it contributes directly to the water-proton spin relaxation process. The poor relaxation efficiency of the cage compound may, however, be improved dramatically by binding the complex to a protein. The efficiency is improved even further if the rotational motion of the protein is reduced drastically by an intermolecular cross-linking reaction. The relaxation efficiency of the cross-linked protein-cage complexes rivals that of the best first-coordination-sphere relaxation agents like [Gd(DTPA)(H2O)]2- and [Gd(DOTA)(H2O)]-.     

Classical molecular dynamics simulation of the photoinduced electron transfer dynamics of plastocyanin.

Classical molecular dynamics simulations are used to investigate the nuclear motions associated with photoinduced electron transfer in plastocyanin. The blue copper protein is modeled using a molecular mechanics potential; potential parameters for the copper-protein interactions are determined using an x-ray crystallographic structure and absorption and resonance Raman spectra. Molecular dynamics simulations yield a variety of information about the ground (oxidized) and optically excited (charge-transfer) states: 1) The probability distribution of the potential difference between the states, which is used to determine the coordinate and energy displacements, places the states well within the Marcus inverted region. 2) The two-time autocorrelation function of the difference potential in the ground state and the average of the difference potential after instantaneous excitation to the excited state are very similar (confirming linear response in this system); their decay indicates that vibrational relaxation occurs in about 1 ps in both states. 3) The spectral densities of various internal coordinates begin to identify the vibrations that affect the optical transition; the spectral density of the difference potential correlation function should also prove useful in quantum simulations of the back electron transfer. 4) Correlation functions of the protein atomic motions with the difference potential show that the nuclear motions are correlated over a distance of more than 20 A, especially along proposed electron transport paths.     

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%.     

Proton magnetic resonance studies of lipid bilayer membranes Experimental determination of inter- and intramolecular nuclear relaxation rates in sonicated phosphatidylcholine bilayer vesicles

We have determined the relative magnitudes of the intra- and intermolecular contributions to the nuclear magnetic relaxation rates of the methylene protons of the hydrocarbon chains in phosphatidylcholine bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). These measurements have been made by the isotopic dilution method using deuterated phosphatidylcholines containing fully deuterated hydrocarbon chains. The results showed that both the methylene linewidths and the spin-lattice relaxation rates are dominated by intramolecular dipolar interactions. Both the intra- and intermolecular contributions to the spin-lattice relaxation rate were found to decrease with increasing temperature and to exhibit a frequency dependence, the rates being higher at the lower NMR frequency in both cases. These observations indicate that both intra- and intermolecular dipolar interactions are modulated by anisotropic motions. In the case of the intermolecular dipolar fields, it is proposed that they are modulated both by the rapid rotational isomerization of the hydrocarbon chains as well as by lateral diffusion of the lipid molecules. That the hydrocarbon chain motion must be fairly effective in effecting efficient spin-lattice relaxation is evident from the negligible intramolecular interchain contribution to the relaxation found in the present work.     

Spin-label studies of the lipid and protein components of erythrocyte membranes. A comparison of electron paramagnetic resonance and saturation transfer electron paramagnetic resonance methods.

We have used both a protein spin label and a lipid spin probe to study some of the slow motions of proteins and of lipids, respectively, in intact erythrocyte membranes. Three electron paramagnetic resonance (EPR) methods, conventional (V1) EPR, second harmonic out-of-phase absorption saturation transfer (ST) EPR (V'2), and first harmonic out-of-phase dispersion ST EPR (U'1) were used to compare the experimental methods and spectral sensitivities with different kinds of molecular motions in human erythrocyte membranes under different experimental conditions. The results show that the V'2 display is relatively more sensitive to the protein motion, while the U'1 display appears more sensitive to the lipid motions, and the V'2 display is substantially more convenient to obtain than the U'1 display.     

Slow motions in chicken villin headpiece subdomain probed by cross-correlated NMR relaxation of amide NH bonds in successive residues

The villin headpiece subdomain (HP36) is a widely used system for protein-folding studies. Nuclear magnetic resonance cross-correlated relaxation rates arising from correlated fluctuations of two N-H^N dipole-dipole interactions involving successive residues were measured at two temperatures at which HP36 is at least 99% folded. The experiment revealed the presence of motions slower than overall tumbling of the molecule. Based on the theoretical analysis of the spectral densities we show that the structural and dynamic contributions to the experimental cross-correlated relaxation rate can be separated under certain conditions. As a result, dynamic cross-correlated order parameters describing slow microsecond-to-millisecond motions of N-H bonds in neighboring residues can be introduced for any extent of correlations in the fluctuations of the two bond vectors. These dynamic cross-correlated order parameters have been extracted for HP36. The comparison of their values at two different temperatures indicates that when the temperature is raised, slow motions increase in amplitude. The increased amplitude of these fluctuations may reflect the presence of processes directly preceding the unfolding of the protein.     

Lipari-Szabo approach as a tool for the analysis of macromolecular gadolinium(III)-based MRI contrast agents illustrated by the [Gd(EGTA-BA-(CH2)12)] n n + polymer

The parameters governing the water proton relaxivity of the [Gd(EGTA-BA-(CH2)12)]nn+ polymeric complex were determined through global analysis of 17O NMR, EPR and nuclear magnetic relaxation dispersion (NMRD) data [EGTA-BA2- = 3,12-bis(carbamoylmethyl)- 6,9-dioxa-3,12-diazatetradecanedioate(2-)]. The Lipari-Szabo approach that distinguishes the global motion of the polymer (tau g) from the local motion of the Gd(III)-water vector (tau l) was necessary to describe the 1H and 17O longitudinal relaxation rates; therefore for the first time it was included in the global simultaneous analysis of the EPR, 17O NMR and NMRD data. The polymer consists on average of only five monomeric units, which limits the intramolecular hydrophobic interactions operating between the (CH2)12 groups. Hence the global rotational correlation time is not very high (tau g298 = 3880 +/- 750 ps) compared to the corresponding DTPA-BA-based polymer (about 15 monomeric units), where tau g298 = 6500 ps. As a consequence, the relaxivity is limited by the rotation, which precludes the advantage obtained from the fast exchanging chelating unit (kex298 = 2.2 +/- 0.1 x 10(6) s-1).     

Proton magnetic relaxation of manganese (II) tetrakis(4-sulfophenyl)porphine ion in water.

Water proton nuclear magnetic spin-lattice relaxation rates are reported as a function of magnetic field strength for aqueous solutions of manganese tetrakis(4-sulfophenyl)porphine complexes. The manganese(III) complex displays relaxation that is remarkably independent of temperature at low magnetic field and a magnetic field dependence that is characteristic of the electron spin relaxation rates, making a contribution to the correlation time that dominates the electron-nuclear coupling. The manganese(II) complex is much more effective in relaxing water protons, but the usual models of first coordination sphere and outer-sphere relaxation fail to account for the magnitude and the magnetic field dependence of the relaxation rates. The data suggest that the delocalization of the electron density into the ligand system provides an increase in the effectiveness of what may be called the outer-sphere paths for water proton relaxation.     

Spin state equilibria in soybean ferric leghemoglobin and its complexes with fomate and acetate

The interactions of fluoride, acetate and formate with soybean ferric leghemoglobin $\text{a}$ have been investigated by ¹H NMR spectroscopy. In the presence of fluoride or acetate leghemoglobin is locked into a high spin ferric conformation whilst the formate complex exists as an equilibrium mixture of high and low spin states. Both formate and acetate ligate directly to the iron and the different magnetic properties of the complexes are attributed to steric constraints within the heme pocket.