Transient deprotonation of bacterial halorhodopsin by photoexcited base

AbstractThe excited singlet state of 6-methoxyquinoline is basic enough to abstract a proton from water and generate OH−. Pulse excitation of such a solution undergoes a massive transient alkalinization. This procedure was employed to monitor the deprotonation of the Schiff base of halorhodopsin. Both the deprotonation and reprotonation of the chromophore are diffusion controlled reactions

The Dynamics of Ca2+ Ions within the Solvation Shell of Calbindin D9k

The encounter of a Ca2+ ion with a protein and its subsequent binding to specific binding sites is an intricate process that cannot be fully elucidated from experimental observations. We have applied Molecular Dynamics to study this process with atomistic details, using Calbindin D9k (CaB) as a model protein. The simulations show that in most of the time the Ca2+ ion spends within the Debye radius of CaB, it is being detained at the 1st and 2nd solvation shells. While being detained near the protein, the diffusion coefficient of the ion is significantly reduced. However, due to the relatively long period of detainment, the ion can scan an appreciable surface of the protein. The enhanced propagation of the ion on the surface has a functional role: significantly increasing the ability of the ion to scan the protein's surface before being dispersed to the bulk. The contribution of this mechanism to Ca2+ binding becomes significant at low ion concentrations, where the intervals between successive encounters with the protein are getting longer. The efficiency of the surface diffusion is affected by the distribution of charges on the protein's surface. Comparison of the Ca2+ binding dynamics in CaB and its E60D mutant reveals that in the wild type (WT) protein the carboxylate of E60 function as a preferred landing-site for the Ca2+ arriving from the bulk, followed by delivering it to the final binding site. Replacement of the glutamate by aspartate significantly reduced the ability to transfer Ca2+ ions from D60 to the final binding site, explaining the observed decrement in the affinity of the mutated protein to Ca2+

Gaugement of the inner space of the apomyoglobin's heme binding site by a single free diffusing proton. II. Interaction with a bulk proton.

The reaction mechanism and the dynamic aspects of protonation of a defined moiety located inside an aqueous cavity in a protein were monitored by time resolved spectroscopy using the pyranine apomyoglobin complex as a model (Shimoni, Tsfadia, Nachliel, and Gutman, 1993, Biophys. J. 64:472-479). The reaction was synchronized by a short laser pulse and the reprotonation of the ground state pyranine anion (phi O-) was monitored, in the microsecond time scale, by its transient absorption at 457 nm. The observed signal was reconstructed by a numeric solution of nonlinear, coupled differential equations which account for the direct reaction of phi O- with bulk proton and by proton transfer from the nearby amino acids: His 64, Asp 44, Asp 60, and Glu 59. A unique combination of rate constant was obtained which quantitates the contribution of each pathway to the overall relaxation process. In the first phase of the dynamics phi O- abstracts a proton from the nearby protonated histidine. The bulk proton interacts preferentially with the cluster of three carboxylates and immediately shuttled to the deprotonated histidine. The high proximity of the reactive groups and the strong electrostatic forces operating inside the heme binding cavity render the rate of proton transfer in the site ultrafast

Dynamic studies of proton diffusion in mesoscopic heterogeneous matrix: II. The interbilayer space between phospholipid membranes

The thin water layer, as found in chloroplast or mitochondria, is confined between low dielectric amphypathic surfaces a few nm apart. The physical properties of this mesoscopic space, and how its dimensions affect the rate of chemical reactions proceeding in it, is the subject for this study. The method selected for this purpose is time resolved fluorometry which can monitor the reversible dissociation of a proton from excited molecule of pyranine (8 hydroxy pyrene 1,3,6 tri sulfonate) trapped in thin water layers of a multilamellar vesicle made of neutral or slightly charged phospholipids. The results were analyzed by a computer program of N. Agmon (Pines, E., D. Huppert, and N. Agmon. 1988. J. Am. Chem. Soc. 88:5620-5630) that simulates the diffusion of a proton, subjected to electrostatic attraction, in a thin water layer enclosed between low affinity, proton binding surfaces. The analysis determines the diffusion coefficient of the proton, the effective dielectric constant of the water and the water accessibility of the phosphomoieties of the lipids. These parameters were measured for various lipids [egg-phosphatidylcholine (egg PC), dipalmitoyl phosphatidylcholine (DPPC), cholesterol + DPPC (1:1) and egg PC plus phosphatidyl serine (9:1)] and under varying osmotic pressure which reduces the width of the water layer down to ∼10 ∼ across. We found that: (a) The effective dielectric constant of the aqueous layer, depending on the lipid composition, is ∼40. (b) The diffusion coefficient of the proton in the thin layer (30-10 ∼ across) is that measured in bulk water D = 9.3 10(-5) cm(2)/s, indicating that the water retains its normal liquid state even on contact with the membrane. (c) The reactivity of the phosphomoiety, quantitated by rate of its reaction with proton, diminishes under lateral pressure which reduces the surface area per lipid. We find no evidence for abnormal dynamics of proton transfer at the lipid water interface which, by any mechanism, accelerates its diffusion

Dynamic studies of proton diffusion in mesoscopic heterogeneous matrix: I. Concentrated solutions of sucrose

Biochemical systems lose their homogeneity at a mesoscopic scale; physical parameters vary sharply over a scale of a few nanometers. In this manuscript, we demonstrate how proton diffusion studies can report the microscopic properties of inhomogeneous systems. The method used for this purpose was the laser induced proton pulse and the reaction followed was the recombination of a proton with pyranine anion (8 hydroxy pyrene 1,3,6 trisulfonate) either in the excited state (subnanosecond dynamics) or in the ground state (microsecond time-scale measurements). The observed signals were analyzed by numeric integration of differential rate equations pertinent to the diffusion controlled reaction between proton and pyranine anion. The accuracy of the methodology was verified by measuring the dielectric constant of sucrose solutions. The results we obtained are identical with those published in the International Critical Tables (1933. Vol. VI, 82-101). The diffusion coefficient of proton was found to be independent of the sucrose concentration, up to 2M solution where the sucrose makes up 45% of the volume. This observation is interpreted in terms of the microscopic heterogeneity of the solution: the proton diffuses in the aqueous space between the sucrose molecules, while the continuity of the aqueous phase is maintained by the Brownian motion of the sucrose molecule, which allows the proton to pass between them at an unhindered rate

Gaugement of the inner space of the apomyoglobin's heme binding site by a single free diffusing proton. I. Proton in the cavity.

Time resolved fluorimetry was employed to monitor the geminate recombination between proton and excited pyranine anion locked, together with less than 30 water molecules, inside the heme binding site of Apomyoglobin (sperm whale). The results were analyzed by a numerical reconstruction of the differential rate equation for time-dependent diffusion controlled reaction with radiating boundaries using N. Agmon's procedure (Huppert, Pines, and Agmon, 1990, J. Opt. Soc. Am. B., 7:1541-1550). The analysis of the curve provided the effective dielectric constant of the proton permeable space in the cavity and the diffusion coefficient of the proton. The electrostatic potential within the cavity was investigated by the equations given by Gilson et al. (1985, J. Mol. Biol., 183:503-516). According to this analysis the dielectric constant of the protein surrounding the site is epsilon prot < or = 6.5. The diffusion coefficient of the proton in the heme binding site of Apomyoglobin-pyranine complex is D = 4 x 10(-5) cm2/s. This value is approximately 50% of the diffusion coefficient of proton in water. The lower value indicates enhanced ordering of water in the cavity, a finding which is corroborated by a large negative enthropy of binding delta S0 = -46.6 cal.mole-1 deg-1. The capacity of a small cavity in a protein to retain a proton had been investigated through the mathematical reconstruction of the dynamics. It has been demonstrated that Coulombic attraction, as large as delta psi of energy coupling membrane, is insufficient to delay a free proton for a time frame comparable to the turnover time of protogenic sites

Time-resolved protonation dynamics of a black lipid membrane monitored by capacitative currents

The laser-induced proton pulse (Gutman, M. (1986) Methods Enzymol. 127, 522-538) was used for transient protonation of one side of a black lipid membrane. The charging of the membrane drives an electric (voltage or current) signal selectively representing the fast proton exchange at the membrane/electrolyte interface. The sensitivity of the electric signal to the presence of buffer indicates that proton transfer is measured, not some dyes or membrane photoelectric artifact. The same event can be visualized in an analogous system consisting of a pH indicator adsorbed to neutral detergent-phospholipid mixed micelles. The time-resolved light absorption transient is equivalent to the electrically determined transient charging of the membrane surface. The sensitivity of the current measurement exceeds the spectrophotometric method by 6-8 orders of magnitudes. As little as 10-18 mol of H+ reacting with 0.75 mm2 of the membrane surface can be monitored in a time-resolved observation. Both types of observed transients were accurately reconstructed by the numerical solution of coupled, non-linear, differential equations describing the system. The rate constants of the various proton transfer reactions were calculated and found to be of diffusion controlled reactions. There is no evidence for any barrier at the interface which either prevents protons from reaching the membrane, or keeps proton on the interface. The electric measurements can be applied for monitoring proton transfer kinetics of complex biomembrane preparations