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At least 127 records · Page 7Linked to original sources
PubMed1984

Proton-sulfate cotransport: external proton activation of sulfate influx into human red blood cells.

Sulfate influx into human red blood cells was measured at 0 and 22 degrees C at several fixed external pH values between 3 and 10. These cells had normal internal pH and chloride concentrations so that sulfate influx was not limited by the efflux half-cycle reactions. The flux was a Michaelis-Menten function of sulfate concentration at each pH with K1/2SO4 = 4-10 mM. External protons activated influx 100-fold at a single site with a pK = 5.9 at 22 degrees C and 5.5 at 0 degrees C. This pK is similar to the value 5.99 +/- 0.3 for external proton binding to the sulfate-loaded transporter at 0 degrees C (J. Gen. Physiol. 79: 87-114, 1982). The flux was stilbene sensitive even in valinomycin-treated cells and was independent of membrane potential. This proton-activated influx appears to be proton-sulfate cotransport. At high pH there was a proton-independent flux that was membrane potential and stilbene sensitive. This proton-insensitive flux appears to be SO4(2-)/Cl- exchange or net sulfate influx. The sulfate influx over the entire pH range may be described in terms of an equation for the sum of the influxes through these two pathways on band 3.

Anion Transport Proteins↗
PubMed1993

Contributions from hydration of carboxylate groups to the spectrum of water-polypeptide proton-proton Overhauser effects in aqueous solution.

Nuclear Overhauser effects (NOE) were measured between water protons and protons of the glutamic acid side chain of the bicyclic decapeptide c-[Arg-Arg-Cys-Asn-Ala-Glu-Ala-Cys-Lys-Gly] in aqueous solution. Positive NOEs were observed between the gamma CH2 group of Glu and the water resonance, with similar NOE intensities at pH 2.0 and pH 6.3 in both the laboratory frame and the rotating frame of reference. These results indicate that the residence times of the hydration water molecules near the side-chain methylene protons are shorter than 500 ps for both the charged form and the uncharged form of Glu, and hence comparable to the water residence times near uncharged amino acid side chains. Furthermore, this study shows that the acidic proton in protonated carboxylic acid groups is not likely to interfere with the observation of polypeptide-hydration water NOEs, which is in contrast to the hydroxyl protons of the side chains of serine, threonine and tyrosine.

Algorithms↗
PubMed1979

Individual assignments of amide proton resonances in the proton NMR spectrum of the basic pancreatic trypsin inhibitor.

Studies of proton-proton nuclear Overhauser effects were used to obtain individual assignments of 17 amide proton resonances in the 360 MHz proton nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor. First, optimizing the conditions for obtaining selective nuclear Overhauser effects in the presence of spin diffusion in macromolecules is discussed. Truncated driven nuclear Overhauser experiments were used to assing the amide proton resonances of the beta-sheet in the inhibitor. It is suggested that these techniques could serve quite generally to obtain individual resonance assignments in beta-sheet secondary structures of proteins. Combination of nuclear Overhauser studies with spin decoupling further resulted in individual assignments of the gamma-methyl resonances of the two isoleucines and numerous Calpha and Cbeta protons.

Amides↗
PubMed1996

Asp76 is the Schiff base counterion and proton acceptor in the proton-translocating form of sensory rhodopsin I.

Both sensory rhodopsin I, a phototaxis receptor, and bacteriorhodopsin, a light-driven proton pump, have homologous residues which have been identified as critical for bacteriorhodopsin functioning. This includes Asp76, which in the case of bacteriorhodopsin (Asp85) functions as both the Schiff base counterion and the proton acceptor. Sensory rhodopsin I exists in a pH dependent equilibrium between two different forms in the absence of its transducer protein HtrI. At pH below 7, it exists primarily in a blue form (lambda max = 587 nm) which functions as a phototaxis signal transducer when complexed to HtrI, while at higher pH, it converts to a purple proton-transporting form similar to bacteriorhodopsin (lambda max = 550 nm). We report ATR-FTIR difference spectra obtained from both low- and high-pH forms of purified sensory rhodopsin I reconstituted into lipid vesicles. The low-pH species has an ethylenic C = C stretch mode at 1520 cm-1 which shifts to 1526 cm-1 in the high-pH form. No frequency shift was found for the mutant D76N, in agreement with visible absorption measurements. Weak negative/positive bands at 1763/1751 cm-1 previously assigned to a perturbation of the C = O stretch mode of Asp76 during S373 formation in the low-pH form are replaced by a single intense positive band near 1749 cm-1 in the high-pH form. These results along with the effects of H/D exchange show that Asp76 is protonated in the signal-transducing form of sensory rhodopsin I and is ionized and functions as the counterion and Schiff base proton acceptor in the proton-transporting high-pH form of sensory rhodopsin I similar to bacteriorhodopsin.

Aspartic Acid↗
PubMed2001

Theoretical study of electron, proton, and proton-coupled electron transfer in iron bi-imidazoline complexes.

A comparative theoretical investigation of single electron transfer (ET), single proton transfer (PT), and proton-coupled electron transfer (PCET) reactions in iron bi-imidazoline complexes is presented. These calculations are motivated by experimental studies showing that the rates of ET and PCET are similar and are both slower than the rate of PT for these systems (Roth, J. P.; Lovel, S.; Mayer, J. M. J. Am. Chem. Soc. 2000, 122, 5486). The theoretical calculations are based on a multistate continuum theory, in which the solute is described by a multistate valence bond model, the transferring hydrogen nucleus is treated quantum mechanically, and the solvent is represented as a dielectric continuum. For electronically nonadiabatic electron transfer, the rate expressions for ET and PCET depend on the inner-sphere (solute) and outer-sphere (solvent) reorganization energies and on the electronic coupling, which is averaged over the reactant and product proton vibrational wave functions for PCET. The small overlap of the proton vibrational wave functions localized on opposite sides of the proton transfer interface decreases the coupling for PCET relative to ET. The theory accurately reproduces the experimentally measured rates and deuterium kinetic isotope effects for ET and PCET. The calculations indicate that the similarity of the rates for ET and PCET is due mainly to the compensation of the smaller outer-sphere solvent reorganization energy for PCET by the larger coupling for ET. The moderate kinetic isotope effect for PCET arises from the relatively short proton transfer distance. The PT reaction is found to be dominated by solute reorganization (with very small solvent reorganization energy) and to be electronically adiabatic, leading to a fundamentally different mechanism that accounts for the faster rate.

Journal Article↗
PubMed1981

Affinities of the protonated and non-protonated forms of hyoscine and hyoscine N-oxide for muscarinic receptors of the guinea-pig ileum and a comparison of their size in solution with that of atropine.

1. At 37 degrees C in 0.1 M NaCl the pKa of hyoscine (10 mM) is 7.53; the non-protonated form has about one-tenth of the affinity (log K = 8.58) of the protonated form (log K = 9.58) for muscarine-sensitive receptors of the guinea-pig ileum at 37 degrees C. 2. In the same conditions the pKa of hyoscine N-oxide is 5.78 and the non-protonated form is inactive on the ileum whereas the protonated form is highly active with log K estimated to be 9.9, at least as active as hyoscine methobromide (log K = 9.85). 3. Hyoscine methobromide appears to occupy less space in water than atropine methobromide; hyoscine hydrochloride occupies less space than hyoscyamine hydrochloride: the non-protonated forms are slightly bigger. Hyoscine N-oxide hydrobromide is slightly smaller than hyoscine methobromide but the removal of the proton is accompanied by a reduction in volume, such as is seen with other zwitterions. 4. These differences in volume indicated a reduction in entropy on solution which may allow a greater increase in entropy on binding to receptors and hence greater affinity. The higher activity of hyoscine itself could also be due to the presence of the N-methyl group in the axial position, rather than equatorial as in hyoscyamine or atropine. 5. The different position of the N-methyl group may partly explain why the pKa of hyoscine is 2 units lower than that of hyoscyamine or atropine. It is also probable that the unionized form of hyoscine is stabilized by hydration. 6. Although hyoscine N-oxide is only weakly active at pH 7.6, it is present in a highly active form in the acid environment of the stomach and so might be expected to act selectively at this site.

Animals↗
PubMed2002

F(0) of ATP synthase is a rotary proton channel. Obligatory coupling of proton translocation with rotation of c-subunit ring.

Coupling of proton flow and rotation in the F(0) motor of ATP synthase was investigated using the thermophilic Bacillus PS3 enzyme expressed functionally in Escherichia coli cells. Cysteine residues introduced into the N-terminal regions of subunits b and c of ATP synthase (bL2C/cS2C) were readily oxidized by treating the expressing cells with CuCl(2) to form predominantly a b-c cross-link with b-b and c-c cross-links being minor products. The oxidized ATP synthases, either in the inverted membrane vesicles or in the reconstituted proteoliposomes, showed drastically decreased proton pumping and ATPase activities compared with the reduced ones. Also, the oxidized F(0), either in the F(1)-stripped inverted vesicles or in the reconstituted F(0)-proteoliposomes, hardly mediated passive proton translocation through F(0). Careful analysis using single mutants (bL2C or cS2C) as controls indicated that the b-c cross-link was responsible for these defects. Thus, rotation of the c-oligomer ring relative to subunit b is obligatory for proton translocation; if there is no rotation of the c-ring there is no proton flow through F(0).

Bacillus↗
PubMed1989

CF0, the proton channel of chloroplast ATP synthase. After removal of CF1 it appears in two forms with highly different proton conductance.

The discharge of the flash-induced transmembrane voltage through the exposed proton channel, CF0, of the chloroplast ATP synthase, CF0CF1 was investigated. EDTA treatment of thylakoid membranes exposed approximately 50% of total CF0 by removal of the CF1 counterparts. This greatly accelerated the decay of the transmembrane voltage, as was apparent from electrochromic-absorption changes of intrinsic pigments and by pH-indicating-absorption changes of added dyes. Two decay processes were discernible, one rapid with a typical half-decay time of 2 ms, and a slower one with a half-decay time variable between 20-100 ms. Both were sensitive to CF0 inhibitors, but only the rapid decay process was also inhibited by added CF1. CF1 was effective in surprisingly small amounts, which were significantly lower than those previously removed by EDTA treatment. This finding corroborated our previous conclusion that the rapid decay of the transmembrane voltage was attributable to only a few high-conductance channels among many CF0 molecules, typically in the order of one channel/CF1-depleted EDTA vesicle. Inhibition of photophosphorylation in control thylakoids was measured as function of the concentration of CF0 inhibitors. It was compared with the inhibition of proton conduction through exposed CF0 in EDTA vesicles. Photophosphorylation and proton conduction by the high-conductance form of CF0 were inhibited by the same low inhibitor concentrations. This suggested that the high-conducting form of CF0 with a time-averaged single-channel conductance of 1 pS [Lill, H., Althoff, G. & Junge, W. (1987) J. Membrane Biol. 98, 69-78] represented the proton channel in the integral enzyme, which acted as a low-impedance access from the thylakoid lumen to the coupling site in CF0CF1. The slow decay process was attributed to a majority of low-conductance CF0 channels, i.e. about 50 molecules/vesicle. The conductance of these channels was more than 100-fold lower and they did not compete with the very few highly conducting channels for rebinding of added CF1. The low proton conduction of the majority of exposed CF0 molecules, possibly due to a structural rearrangement, may be protecting the thylakoid membrane against rapid energy dissipation caused by accidental loss of CF1. It may also explain the low single-channel conductance of bacterial F0 reported in the literature.

Binding Sites↗
PubMed1984

Polypeptide secondary structure determination by nuclear magnetic resonance observation of short proton-proton distances.

The use of proton-proton nuclear Overhauser enhancement (NOE) distance information for identification of polypeptide secondary structures in non-crystalline proteins was investigated by stereochemical studies of standard secondary structures and by statistical analyses of the secondary structures in the crystal conformations of a group of globular proteins. Both regular helix and beta-sheet secondary structures were found to contain a dense network of short 1H-1H distances. The results obtained imply that the combined information on all these distances obtained from visual inspection of the two-dimensional NOE (NOESY) spectra is sufficient for determination of the helical and beta-sheet secondary structures in small globular proteins. Furthermore, cis peptide bonds can be identified from unique, short sequential proton-proton distances. Limitations of this empirical approach are that the exact start or end of a helix may be difficult to define when the adjoining residues form a tight turn, and that unambiguous identification of tight turns can usually be obtained only in the hairpins of antiparallel beta-structures. The short distances between protons in pentapeptide segments of the different secondary structures have been tabulated to provide a generally applicable guide for the analysis of NOESY spectra of proteins.

Aprotinin↗
PubMed2004

Water alignment, dipolar interactions, and multiple proton occupancy during water-wire proton transport.

A discrete multistate kinetic model for water-wire proton transport is constructed and analyzed using Monte Carlo simulations. In the model, each water molecule can be in one of three states: oxygen lone-pairs pointing leftward, pointing rightward, or protonated (H(3)O(+)). Specific rules for transitions among these states are defined as protons hop across successive water oxygens. Our model also includes water-channel interactions that preferentially align the water dipoles, nearest-neighbor dipolar coupling interactions, and Coulombic repulsion. Extensive Monte Carlo simulations were performed and the observed qualitative physical behaviors discussed. We find the parameters that allow the model to exhibit superlinear and sublinear current-voltage relationships, and show why alignment fields, whether generated by interactions with the pore interior or by membrane potentials, always decrease the proton current. The simulations also reveal a "lubrication" mechanism that suppresses water dipole interactions when the channel is multiply occupied by protons. This effect can account for an observed sublinear-to-superlinear transition in the current-voltage relationship.

Biological Transport↗
PubMed1978

Amide proton spin-lattice relaxation in polypeptides. A field-dependence study of the proton and nitrogen dipolar interactions in alumichrome.

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the (1)H spinlattice relaxation times (T(1)'s) are distinct in the Al(3+)-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of (1)H-(1)H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T(1) (-1) and Sigmar(-6) at the three magnetic fields. Considering the very sensitive r-dependence of T(1), the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T(1) (-1) is proposed to mainly originate from the (1)H-(14)N dipolar interaction: this was supported by comparison with measurements on an (15)N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {(1)H}-(1)H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the (1)H-(1)H dipolar mechanism to cause NOE's. From the slope and intersect values of T(1) (-1) vs. Sigmar(-6) linear plots, a number of independent estimates of tau(r), the rotational correlation time, were derived. These and the field-dependence of the T(1)'s yield a best estimate approximately 0.37 ns, in good agreement with 0.38 ns [unk] [unk] 0.41 ns, previously determined from (13)C and (15)N spin-lattice relaxation data.

Aluminum↗
PubMed1983

Proton magnetic resonance study of the influence of heme 2,4 substituents on the exchange rates of labile protons in the heme pocket of myoglobin.

Four exchangeable protons with large hyperfine shifts are assigned in the heme pocket of sperm whale met-cyano myoglobin reconstituted with heme possessing acetyl groups, ethyl groups, bromines, and hydrogens at the 2,4 position, using both relaxation and chemical-shift data. The four protons arise from the ring NH's of the proximal (F8), distal (E7), and FG2 histidines, and the peptide NH of His F8. The similarity of all chemical shifts to those of the native protein as well as the invariance of the relaxation rates of the distal histidyl ring NH dictate essentially the same structure for the heme cavity of both native and reconstituted proteins. The exchange rates with bulk water of the four labile proteins in each modified protein were determined by saturation-transfer and line width methods. All four labile protons were found to have the same exchange rate as in the native protein for acetyl and ethyl 2,4 substituents; the two resolved labile protons in the derivative with 2,4 bromine were also unchanged. The reconstituted protein with hydrogens at the 2,4 position exhibited slower exchange rates for three of the four protons, indicating an increased dynamic stability of the heme pocket in the absence of bulky 2,4 substituents.

Animals↗
PubMed1999

Molecular basis for pH sensitivity and proton transfer in green fluorescent protein: protonation and conformational substates from electrostatic calculations.

We performed a theoretical study to elucidate the coupling between protonation states and orientation of protein dipoles and buried water molecules in green fluorescent protein, a versatile biosensor for protein targeting. It is shown that the ionization equilibria of the wild-type green fluorescent protein-fluorophore and the internal proton-binding site E222 are mutually interdependent. Two acid-base transitions of the fluorophore occur in the presence of neutral (physiologic pH) and ionized (pH > 12) E222, respectively. In the pH-range from approximately 8 to approximately 11 ionized and neutral sites are present in constant ratio, linked by internal proton transfer. The results indicate that modulation of the internal proton sharing by structural fluctuations or chemical variations of aligning residues T203 and S65 cause drastic changes of the neutral/anionic ratio-despite similar physiologic fluorophore pK(a) s. Moreover, we find that dipolar heterogeneities in the internal hydrogen-bond network lead to distributed driving forces for excited-state proton transfer. A molecular model for the unrelaxed surrounding after deprotonation is discussed in relation to pathways providing fast ground-state recovery or slow stabilization of the anion. The calculated total free energy for excited-state deprotonation ( approximately 19 k(B)T) and ground-state reprotonation ( approximately 2 k(B)T) is in accordance with absorption and emission data (</=5000 cm(-1) or 24 k(B)T).

Binding Sites↗
PubMed1984

The catalytic mechanism of serine proteases: single proton versus double proton transfer.

The two possible mechanisms of proton transfer on the catalytic process of serine proteases (single or double proton transfer) have been analysed. Intermediate neglect of differential overlap calculations have been performed in the absence and in the presence of the substrate molecule and one water molecule localized in the active site. It is shown that, in the absence of the substrate and water, double proton transfer seems to be the most feasible mechanism. However, when these molecules are introduced in the calculation, the role played by them is to facilitate the formation of the zwitterionic structure (single proton transfer) and to destabilize the intermediate structure which leads to double proton transfer. All calculations were made in vacuo.

Binding Sites↗
PubMed2003

pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates, chromophore deprotonation by hydroxyl uptake, and protonation relaxation of the dark state.

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.

Bacterial Proteins↗