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PubMed2002

Coupled protonic and electronic conduction in the molecular conductor [2-(2-1H-Benzimidazolyl)-1H-benzimidazolium]-TCNQ.

A novel molecular based proton-electron mixed conductor, (H3BBIM(+))(TCNQ)(Cl(-))(0.5)(H(2)O) (1), where H3BBIM(+) is 2-(2-1H-benzimidazolyl)-1H-benzimidazolium and TCNQ is 7,7,8,8-tetracyano-p-quinodimethane, was synthesized. The salt exhibited peculiar phase transitions as a result of proton-electron coupling phenomena within the crystal. Salt 1 is composed of a closed-shell H3BBIM(+) cation and an open-shell TCNQ anion radical, and was obtained by electrocrystallization in a buffered CH(3)CN solution. Crystal 1 was constructed from the segregated uniform stacks of H3BBIM(+) and TCNQ. The regular stack of partially electron-transferred TCNQ(-0.5) provided a one-dimensional electron-conducting column. Between the regular H3BBIM(+) columns, a channel-like sequence of holes was formed at the side-by-side space that is filled with disordered Cl(-) ions and H(2)O molecules, and which offer a proton-conducting path. The electrical conductivity at room temperature (10 S cm(-1)) was greater by a magnitude of four than the protonic conductivity (1x10(-3) S cm(-1)). Electronic conduction changed from metallic (T>250 K) to semiconducting (250>T>100 K), then insulating (T<100 K). Protonic conductivity was observed above 200 K. The continuous metal-semiconductor transition at 250 K is caused by the formation of the Cl(-) superstructure, whereas the disappearance of protonic conductivity at 200 K is related to the rearrangement of the [Cl(-)-(H(2)O)(2)] sublattice within the channel. The magnetic susceptibility continuously shifted from Pauli paramagnetism (T>250 K) to the one-dimensional linear Heisenberg antiferromagnetic chain (T<250 K). Lattice dimerization in regular TCNQ columns was confirmed by the appearance of vibrational a(g) mode at low temperatures. The strong localization of conduction electrons on each TCNQ dimer caused a Mott transition at 100 K. The melting and freezing of the [Cl(-)-(H(2)O)(2)] sublattice within the channel was correlated to the conduction electrons on the TCNQ stack and the protonic conductivity.

Journal Article↗
PubMed2002

Substituent effect and multisite protonation in the fragmentation of alkyl benzoates.

The dissociation of protonated alkyl benzoates (para H, CN, OMe and NO(2)) into protonated benzoic acids and alkyl cations was studied in the gas phase. It was found that the product ratio depends on the substituent at the para position of the phenyl ring. The substituent effect is probably the result of the formation of an ion-neutral complex intermediate that decomposes to an ion and a neutral, according to the relative proton affinities of the two moieties. The experimental results and theoretical calculations indicate that the favored protonation site in these compounds is the ester's carbonyl and that proton transfer from the phenyl ring to the ester group is very likely to occur under chemical ionization conditions. It is most probable that the carbonyl protonated form is a common intermediate in the fragmentation process, regardless of the protonation site.

Journal Article↗
PubMed2005

Isomerization of the protonated acetone dimer in the gas phase.

Mass spectrometry-based methods have been employed in order to study the reactions of non- (h(6)/h(6)), half (d(6)/h(6)), and fully (d(6)/d(6)) deuterium labeled protonated dimers of acetone in the gas phase. Neither kinetic nor thermodynamic isotope effects were found. From MIKES experiments (both spontaneous and collision-induced dissociations), it was found that the relative ion yield (m/z 65 vs m/z 59) from the dissociation reaction of half deuterium labeled (d(6)/h(6)) protonated dimer of acetone is dependent on the internal energy. A relative ion yield (m/z 65 vs m/z 59) close to unity is observed for cold, nonactivated, metastable ions, whereas the ion yield is observed to increase (favoring m/z 65) when the pressure of the collision gas is increased. This is in striking contrast to what would be expected if a kinetic isotope effect were present. A combined study of the kinetics and the thermodynamics of the association reaction between acetone and protonated acetone implicates the presence of at least two isomeric adducts. We have employed G3(MP2) theory to map the potential energy surface leading from the reactants, acetone and protonated acetone, to the various isomeric adducts. The proton-bound dimer of acetone was found to be the lowest-energy isomer, and protonated diacetone alcohol the next lowest-energy isomer. Protonated diacetone alcohol, even though it is an isomer hidden behind many barriers, can possibly account for the observed relative ion yield and its dependence on the mode of activation.

Journal Article↗
PubMed1987

NMR spectroscopy of heterogeneous solid-liquid mixtures. Spin grouping and exchange analysis of proton spin relaxation in a tissue.

An attempt to resolve the complicated proton NMR signal of a heterogeneous material is presented. Lung tissue is used to illustrate the approach to this problem, which is based on resolving the total proton magnetization into components according to their relaxation times. This procedure is utilized at both low and high magnetic fields. These results are then correlated and analyzed for the effect of exchange between spin groups. For example, the total proton NMR signal of lung tissue is shown to arise from three interacting proton groups: protons on solid macromolecules, bound water protons, and bulk water protons. The dynamics of water is modeled to satisfy the dispersivity and the values of the relaxation parameters obtained considering the effect of exchange. It is proposed that this approach is generally applicable.

Animals↗
PubMed2006

Is proton cationization promoted by polyatomic primary ion bombardment during time-of-flight secondary ion mass spectrometry analysis of frozen aqueous solutions?

Ion bombardment of pure water ice by Au+ monoatomic and Au3 + and C60 + polyatomic projectiles results in the emission of two series of water cluster ions-(H2O)n + and (H2O)nH+-with n ranging from 1 to >40. The cluster ion yields are very significantly higher under polyatomic ion bombardment than when using an Au+ primary ion. The yield of the protonated water species (H2O)nH+ is found to be enhanced by increasing ion fluence. C60 + bombardment results in a very dramatic increase in the (H2O)nH+ yield and decrease in the yield of (H2O)n +. Au3 + also significantly increased the yield of protonated species relative to the non-protonated but to a lesser extent than C60 +. Bombardment by Au+ also increased the yield of protonated species but to a very much smaller extent. The hypothesis that the protonated species may enhance the yield of [M+H]+ from solute molecules in solution has been investigated using two amino acids, alanine and arginine, and a nucleic base, adenine. The data suggest that the protons produced by the sputtering of water ice are depleted in the presence of these solutes and concurrently the yields of solute-related [M+H]+ and immonium secondary ions are greatly enhanced. These yield enhancements are analysed in the light of other possible contributors such as increased rates of sputtering under polyatomic beams and increased secondary ion yields as a consequence of solute dispersion. It is concluded that enhanced proton attachment is occurring in polyatomic sputtered frozen aqueous solutions.

Journal Article↗
PubMed1996

Intrinsic Proton Affinity of Reactive Surface Groups of Metal (Hydr)oxides: The Bond Valence Principle

The proton affinity of individual surface groups has been calculated with a redefined version of the multi site complexation (MUSIC) model. In the new approach the proton affinity of an oxygen originates from the undersaturation of the oxygen valence. The factors valence and coordination number, which are the basis of Pauling's definition of bond valence, in combination with the number of coordinating (Me and H) ions, are dominant in determining the proton affinity. The neutralization of an oxygen by Me ion(s) is calculated on the basis of the actual bond valence, which accounts for structural details, resulting from an asymmetrical distribution of charge in the coordination environment. An important role in the new version of the MUSIC model is given to the H bonds. The model shows that the proton affinity is determined not only by the number of donating H bonds but also by the number of accepting H bonds. The proton affinity of surface groups and of solution complexes can be understood in one theoretical framework, on the basis of a different number of donating and accepting H bonds. The MUSIC model predicts the variation in proton affinity constants for surface groups in particular those with the same number of coordinating Me ions but with a different structural position. The model is able to predict on the basis of the proton affinity of the individual groups the correct PZC of Me hydroxides, oxohydroxides, and oxides, and explains previous exceptions. The model can also be applied in general to other minerals.

Journal Article↗
PubMed1996

Line Narrowing in Solid-State Proton NMR with Acquisition Delay

Organic solids have extensive proton-proton dipolar interactions, and their 1H NMR linewidths are very large even with magic-angle spinning at moderate speeds. Recently it has been shown that substantial narrowing of the proton linewidths of organic solids can be achieved by using single-pulse excitation with acquisition delay or spin echo [S. Ding and C. A. McDowell, J. Magn. Reson. A 111, 212 (1994); 115, 141 (1995); 117, 171 (1995)]. This interesting line-narrowing phenomenon has been further examined through the study of several amino acids, their deuterated analogs, and some aromatic compounds. The results confirm that narrow proton peaks are observed with long acquisition delay, and the peaks appear in the appropriate chemical-shift ranges for organic protons (0-10 ppm with respect to tetramethylsilane). However, except for some special cases, the observed peaks cannot be assigned to individual types of protons based on chemical-shift considerations only. To explore the reason for the line narrowing, the effect of acquisition delay on the 19F linewidth of CaF2 was also studied and compared with that on the 1H linewidths of organic solids. It is suggested that the broad proton peak in an organic solid is a superposition of numerous transitions. These transitions have different linewidths, and the narrow peaks in the spectrum remain observable with long acquisition delays.

Journal Article↗
PubMed1976

On the mechanism of action of oligomycin and acidic uncouplers on proton translocation and energy transfer in "sonic" submitochondrial particles.

A study is presented of the effect of acidic uncouplers and oligomycin on energy-linked and passive proton translocation, oxidative phosphorylation, and energy-linked nicotinamide-adenine-nucleotide transhydrogenase in EDTA submitochondrial particles from beef-heart. A flow potentiometric technique has been applied to resolve the kinetics of the initial rapid phase of the redox proton pump. Rapid kinetics analysis shows that carbonyl-cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) does not exert any direct effect on redox-linked active proton transport. The uncoupling action of FCCP on oxidative phosphorylation and energy-linked transhydrogenase is shown to be quantitatively accounted for by its promoting effect of passive proton-diffusion across the mitochondrial membrane. Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase. In fact, rapid kinetic analysis demonstrates that oligomycin does not directly affect the redox-linked proton pump. The present results show that there does not exist any labile intermediate in the redox-linked proton pump which is sensitive to acidic uncouplers.

Animals↗
PubMed2006

An automated assignment-free Bayesian approach for accurately identifying proton contacts from NOESY data.

The identification of proton contacts from NOE spectra remains the major bottleneck in NMR protein structure calculations. We describe an automated assignment-free system for deriving proton contact probabilities from NOESY peak lists that can be viewed as a quantitative extension of manual assignment techniques. Rather than assigning contacts to NOESY crosspeaks, a rigorous Bayesian methodology is used to transform initial proton contact probabilities derived from a set of 2992 protein structures into posterior probabilities using the observed crosspeaks as evidence. Given a target protein, the Bayesian approach is used to derive probabilities for all possible proton contacts. We evaluated the accuracy of this approach at predicting proton contacts on 60 (15)N separated NOESY and (13)C separated NOESY datasets simulated from experimentally determined NMR structures and compared it to CYANA, an established method for proton constraint assignment. On average, at the highest confidence level, our method accurately identifies 3.16/3.17 long range contacts per residue and 12.11/12.18 interresidue proton contacts per residue. These accuracies represent a significant increase over the performance of CYANA on the same data set. On a difficult real dataset that is publicly available, the coverage is lower but our method retains its advantage in accuracy over CANDID/CYANA. The algorithm is publicly available via the Protinfo NMR webserver http://protinfo.compbio.washington.edu/protinfo_nmr .

Algorithms↗
PubMed1976

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane.

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side. In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21degreesC. In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds. Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds. The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.

Bacteriorhodopsins↗
PubMed1982

Efficiency of proton extrusion by chemically modified mitochondria.

Bovine heart mitochondria were treated with limited amounts of iodoacetamide, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, phenylglyoxal, tetranitromethane and 1-fluoro-2,4-dinitrobenzene, respectively. Examination of the respiration and proton extrusion characteristics of the chemically modified mitochondria suggests that sulfhydryl and imidazole groups are not directly involved in proton pumping, but that some of the labeled carboxyl, amino, guanidinium and phenolic groups may participate in an indirect proton-extrusion process. Cross-linking mitochondria with glutaraldehyde drastically decreases the efficiency of proton extrusion, whereas treatment of mitochondria with valeraldehyde under similar conditions did not affect the proton-pumping efficiency significantly. The latter observations show that conformational change in the inner mitochondrial membrane may play a crucial role in the active translocation of protons coupled to electron transport. Comparison of the reactivities of the essential amino and carboxyl groups in mitochondria in different oxidation states suggests that these two types of essential functional groups are more exposed to water in the oxidized state. An indirect mechanism for proton pumping based on protein conformational change driven by electron transport based on the results of the present chemical modification studies is suggested.

Animals↗
PubMed1984

Trans to cis proton concentration gradients accelerate ionophore A23187-mediated net fluxes of Ca2+ across the human red cell membrane.

Ionophore A23187-mediated net influx of Ca2+ in ATP-depleted human red cells was studied as a function of the pH and the proton concentration gradient across the membranes. Utilizing the Ca2+-induced increase in K+ conductance of the cell membranes, various CCCP-mediated proton gradients were raised across the membranes of cells suspended in unbuffered salt solutions with different K+ concentrations. In ionophore-mediated equilibrium the concentration ratios of ionized Ca between ATP-depleted, DIDS-treated cells and their suspension medium were equal to the concentration ratios of protons raised to the second power. With no proton concentration gradient across the membranes the net influxes of Ca2+ as a function of pH resembled a titration curve of a weak acid, with half maximal net influx at pH 7.3, at 100 microM extracellular Ca2+. With cellular pH fixed at various values, the net influx of Ca2+ was determined as a function of the proton concentration gradient. A linear relationship between the logarithm of net influx and the difference between extracellular and cellular pH was found at all cellular pH values tested, but the proton concentration gradient acceleration was a function of the cellular pH. Accelerations between 10- and 40- times per unit delta pH were found and net effluxes were correspondingly decreased. The results are discussed in relation to present models of the mechanism of ionophore A23187-mediated Ca2+ transport. The importance of the proton concentration gradient dependency is discussed in relation to the induced oscillations in K+-conductance of human red cell membranes previously reported (Vestergaard-Bogind and Bennekou (1982) Biochim. Biophys. Acta 688, 37-44).

Calcimycin↗
PubMed1987

A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin.

High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated alpha or beta subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfield from H2O to the hydrogen-bonded proton between alpha 103(G10)His and beta 108(G10)Asn at the alpha 1 beta 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H2O previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformational effects of single amino-acid substitutions in mutant hemoglobin molecules.

Adult↗
PubMed2005

The proton nuclear magnetic shielding tensors in biphenyl: experiment and theory.

Line-narrowing multiple pulse techniques are applied to a spherical sample crystal of biphenyl. The 10 different proton shielding tensors in this compound are determined. The accuracy level for the tensor components is 0.3 ppm. The assignment of the measured tensors to the corresponding proton sites is given careful attention. Intermolecular shielding contributions are calculated by the induced magnetic point dipole model with empirical atom and bond susceptibilities (distant neighbours) and by a new quantum chemical method (near neighbours). Subtracting the intermolecular contributions from the (correctly assigned) measured shielding tensors leads to isolated-molecule shielding tensors for which there are symmetry relations. Compliance to these relations is the criterion for the correct assignment. The success of this program indicates that intermolecular proton shielding contributions can be calculated to better than 0.5 ppm. The isolated-molecule shielding tensors obtained from experiment and calculated intermolecular contributions are compared with isolated-molecule quantum chemical results. Expressed in the icosahedral tensor representation, the rms differences of the respective tensor components are below 0.5 ppm for all proton sites in biphenyl. In the isolated molecule, the least shielded direction of all protons is the perpendicular to the molecular plane. For the para proton, the intermediate principal direction is along the C-H bond. It is argued that these relations also hold for the protons in the isolated benzene molecule.

Algorithms↗
PubMed1980

Hydrogen isotope exchange kinetics of single protons in bovine pancreatic trypsin inhibitor.

The exchange kinetics of the slowest exchanging BPTI beta-sheet protons are complex compared to model peptides; the activation energy, E alpha, and the pH dependence are temperature dependent. We have measured the exchange kinetics in the range pH 1--11, 33--71 degrees C, particularly the temperature dependence. The data are fit to a model in which exchange of each proton is determined by two discrete dynamical processes, one with E alpha approximately 65 kcal/mol and less than first order dependence on catalyst ion, and one with E alpha 20--30 kcal/mol and approaching first order in catalyst ion. The low activation energy process is the mechanism of interest in the native conformation of globular proteins and involves low energy, small amplitude fluctuations; the high activation energy process involves major unfolding. The model is simple, has a precedent in the hydrogen exchange literature, and explains quantitatively the complex feature of the exchange kinetics of single protons in BPTI, including the following. For the slowest exchanging protons, in the range 36 degrees--68 degrees C, E alpha is approximately 65 kcal/mol at pH approximately 4, 20--30 kcal/mol at pH greater than 10, and rises to approximately 65 kcal/mol with increasing temperature at pH 6--10; the Arrhenius plots converge around 70 degrees C; the pH of minimum rate, pHmin, is greater than 1 pH unit higher at 68 degrees C than for model compounds; and at high pH, the pH-rate profiles shift to steeper slope; the exchange rates around pHmin are correlated to the thermal unfolding temperature in BPTI derivatives (Wagner and Wüthrich, 1979, J. Mol. Biol. 130:31). For the more rapidly exchanging protons in BPTI the model accounts for the observation of normal pHmin and E alpha of 20--30 kcal/mol at all pH's. The important results of our analysis are (a) rates for exchange from the folded state of proteins are not correlated to thermal lability, as proposed by Wuthrich et al. (1979, J. Mol. Biol. 134:75); (b) the unfolding rate for the BPTI cooperative thermal transition is equal to the observed exchange rates of the slowest exchanging protons between pH 8.4--9.6, 51 degrees C; (c) the rates for exchange of single protons from folded BPTI are consistent with our previous hydrogen-tritium exchange results and with a penetration model of the dynamic processes limiting hydrogen exchange.

Animals↗
PubMed1998

The coupling of electron transfer and proton translocation: electrostatic calculations on Paracoccus denitrificans cytochrome c oxidase.

We have calculated the electrostatic potential and interaction energies of ionizable groups and analyzed the response of the protein environment to redox changes in Paracoccus denitrificans cytochrome c oxidase by using a continuum dielectric model and finite difference technique. Subsequent Monte Carlo sampling of protonation states enabled us to calculate the titration curves of all protonatable groups in the enzyme complex. Inclusion of a model membrane allowed us to restrict the calculations to the functionally essential subunits I and II. Some residues were calculated to have complex titration curves, as a result of strong electrostatic coupling, desolvation, and dipolar interactions. Around the heme a3-CuB binuclear center, we have identified a cluster of 18 strongly interacting residues that account for most of the proton uptake linked to electron transfer. This was calculated to be between 0.7 and 1.1 H+ per electron, depending on the redox transition considered. A hydroxide ion bound to CuB was determined to become protonated to form water upon transfer of the first electron to the binuclear site. The bulk of the protonation changes linked to further reduction of the heme a3-CuB center was calculated to be due to proton uptake by the interacting cluster and Glu(II-78). Upon formation of the three-electron reduced state (P1), His325, modeled in an alternative orientation away from CuB, was determined to become protonated. The agreement of these results with experiment and their relevance in the light of possible mechanisms of redox-coupled proton transfer are discussed.

Catalysis↗
PubMed2000

An algorithm for predicting proton nuclear magnetic resonance deshielding over a carbon-carbon double bond.

Hydrogen nuclei located over a carbon-carbon double bond in a strong magnetic field experience NMR shielding effects that result from the magnetic anisotropy of the nearby double bond and various other intramolecular shielding effects. We have used GIAO, a subroutine in Gaussian 98, to calculate isotropic shielding values and to predict the proton NMR shielding increment for a simple model system: methane held in various orientations, positions, and distances over ethene. The average proton NMR shielding increments of several orientations of methane have been plotted versus the Cartesian coordinates of the methane protons relative to the center of ethene. A single empirical equation for predicting the NMR shielding experienced by protons over a carbon-carbon double bond has been developed from these data. The predictive capability of this equation has been validated by comparing the shielding increments for several alkenes calculated using our equation to the experimentally observed shielding increments. This equation predicts the NMR shielding effects more accurately than previous models that were based on fewer geometries of methane over ethene. In fact, deshielding is predicted by this equation for protons over the center and within about 3 A of a carbon-carbon double bond. This result is in sharp contrast to predictions made by the long-held McConnell "shielding cone" model found in nearly every textbook on NMR, but is consistent with experimental observations. The algorithm for predicting the (de)shielding increment for a proton over an alkene can be used in a spreadsheet on a PC or incorporated into software that estimates chemical shifts using additive substituent constants or a database of structures. In either application its use can substantially improve the accuracy of the estimated chemical shift of a proton in the vicinity of a carbon-carbon double bond, and thus assist in spectral assignments and in correct structure determination.

Algorithms↗
PubMed1995

NMR behavior of the aromatic protons of bovine neurophysin-I and its peptide complexes: implications for solution structure and for function.

The NMR behavior of the aromatic protons of bovine neurophysin-I and its complexes was interpreted with reference to the 2.8 A crystal structure of the dipeptide complex of bovine neurophysin-II and to mechanisms underlying the thermodynamic linkage between neurophysin dimerization and peptide binding. Large binding-induced shifts in the ring proton signals of Tyr-2 of ligand peptides (approximately 0.5 ppm upfield and approximately 0.35 ppm downfield at 25 degrees C for the 3,5- and 2,6-ring protons, respectively) were demonstrated. Consistent with the crystal structure, and in disagreement with conclusions by other investigators, evidence is presented indicating the absence of dipolar contact between Tyr-2 ring protons and protein Phe ring protons. The large binding-induced shifts are attributed to a strong influence of proximal neurophysin carbonyl and disulfide groups on the bound Tyr-2 ring, of potential importance in binding specificity. Resolution of the behavior of neurophysin Phe residues -22 and -35 and of their proton NOE contacts provided insights into the conformational changes associated with peptide binding and with dimerization. Within the amino domain of the protein, as evidenced by the behavior of interface residue Phe-35 and its NOE contacts, binding-induced changes in the subunit interface appeared to involve principally the junction between this interface region and the 3,10-helix that connects it to the binding site in the liganded state. By contrast, as judged by the NOE contacts of His-80, the corresponding interface participant of the carboxyl domain, peptide binding induced a marked decrease in side-chain mobility within the carboxyl domain segment of the interface. Interactions of Phe-22 with protons assigned to Ala-68, neither of which is an interface participant, were demonstrated to be markedly altered both by dimerization in the unliganded state and by peptide binding to the dimer. Since Phe-22 is adjacent to the peptide-binding site, the results collectively support a model in which conformational differences between unliganded monomer and dimer are important contributors to the preferential binding of peptide to the dimer and indicate that the amino and carboxyl domain segments of the interface, which are homologous, are affected differently by peptide binding.

Animals↗