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M P Heyn

Publications and source records attributed to M P Heyn.

At least 55 records · Page 3Linked to original sources
PubMed1989

Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin.

Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (tau)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.

Aspartic Acid↗
PubMed1989

Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement.

The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase and release and uptake of protons from the aqueous phase were investigated for bacteriorhodopsin mutants with aspartic acid-96 replaced by asparagine or glutamic acid. At neutral pH the main effect of the Asp-96----Asn mutation is to slow by 2 orders of magnitude the decay of the M intermediate and the concomitant charge displacement associated with the reprotonation of the Schiff base from the cytoplasmic side of the membrane. The proton uptake measured with the indicator dye pyranine is likewise slowed without affecting the stoichiometry of proton pumping. The corresponding results for the Asp-96----Glu mutant, on the other hand, are very close to those for the wild-type protein. These results provide a kinetic explanation for the fact that at pH 7 and saturating light intensities the steady-state proton pumping is almost abolished in the Asp-96----Asn mutant but is close to normal in the Asp-96----Glu mutant. Thus, the pump is simply turning over much more slowly in the Asp-96----Asn mutant. The time constants of the decay of M and the associated charge translocation increase strongly with increasing pH for the Asp-96----Asn mutant but are virtually pH-independent for the Asp-96----Glu mutant and wild-type bacteriorhodopsin. At pH 5 the M decay of the Asp-96----Asn mutant is as fast as for wild type. These results suggest that Asp-96 serves as an internal proton donor in the proton-uptake pathway from the cytoplasm to the Schiff base.

Asparagine↗
PubMed1988

High-sensitivity neutron diffraction of membranes: Location of the Schiff base end of the chromophore of bacteriorhodopsin.

THREE IMPORTANT EVENTS IN THE FUNCTIONAL CYCLE OF BACTERIORHODOPSIN OCCUR AT THE CHROMOPHORE: the primary absorption of light, the isomerization from the alltrans to the 13-cis form, and the deprotonation and reprotonation of its Schiff base. The protonated Schiff base linkage of the chromophore with lysine-216 plays an essential role in the color regulation of the pigment and is most likely directly involved in the charge translocation of this light-driven proton pump. Although much is known about the structure of the protein, the position of this key functional group has not yet been determined. We have synthesized a retinal in which the five protons closest to the Schiff base are replaced by deuterons. The labeled retinal was spontaneously incorporated into bacteriorhodopsin by using a mutant of Halobacterium halobium that is deficient in the synthesis of retinal. The position of the labeled Schiff base end of the chromophore was determined in the two-dimensional projected density of dark-adapted bacteriorhodopsin by neutron diffraction. The result fits very well with our previous work using retinals that were selectively deuterated in the middle of the polyene chain or in the cyclohexene ring. A coherent structure emerges with the three labeled positions on one line, separated by distances that are in good agreement with the tilt angle of the polyene chain (about 20 degrees ). The chromophore is located in the interior of the protein with the nitrogen of the Schiff base between helices 2 and 6 and with its ring in the vicinity of helix 4. Our results show that it is possible to locate a small group containing as few as five deuterons in a membrane protein of molecular weight 27,000.

Journal Article↗
PubMed1986

Location of the cyclohexene ring of the chromophore of bacteriorhodopsin by neutron diffraction with selectively deuterated retinal.

We report on the location of the cyclohexene ring of the retinylidene chromophore of bacteriorhodopsin projected onto the plane of the membrane. For this purpose, partially deuterated retinal was synthesized containing 11 deuterons at the following positions of the cyclohexene ring: one at C-2, two at C-4, three at C-16, three at C-17, and two at C-18. The partially deuterated retinal was incorporated biosynthetically during growth of the bacteria by using the mutant JW5, which is deficient in the synthesis of retinal. Undeuterated samples were prepared in the same way. Characterization by x-ray diffraction and absorption spectroscopy showed that these samples are identical to native purple membranes as judged by these criteria. A Fourier difference map was calculated from the differences in in-plane diffraction intensities between the deuterated and undeuterated dark-adapted membrane samples. Model calculations showed that the observed difference density had the amplitude expected for a label containing 11 deuterons. At 8.7 A resolution, the map shows one major peak with the center of mass of the deuterated ring in the interior of the molecule between helices 3, 4, 5, and 6. Based on this result and on our previous work on the location of the middle of the polyene chain, we conclude that the COOH-terminal helix G, to which retinal is attached at lysine-216, is either helix 2 or helix 6.

Journal Article↗
PubMed1985

Covalent labeling of functional states of the acetylcholine receptor. Effects of antagonists on the receptor conformation.

Photoaffinity labeling of membrane-bound nicotinic acetylcholine receptor from Torpedo marmorata electric tissue with the ion-channel blocker [3H]TPMP+ reveals various functional states of the receptor protein if labeling is performed with ms time resolution. In the resting and in the activated state most of the label is incorporated into the alpha-polypeptide chains of the receptor complex. When equilibrated with agonists and antagonists, predominantly the delta-polypeptide chain (and to a lesser extent the beta-chain) reacts with the photolabel. Reactivity of the delta-chain increases after exposure to cholinergic effectors with a half-life slower than the kinetics of receptor activation or rapid desensitization. Agonists and antagonists stimulate photolabelling of the delta-chain with different kinetics. For acetylcholine, carbamoylcholine and suberyldicholine the half-life of the reactivity increases is 400 - 500 ms; for the antagonists hexamethonium, d-tubocurarine and flaxedil it is about 10 s. The latter slow kinetics are also observed when the receptor is preequilibrated with agonists or antagonists prior to mixing with [3H]TPMP+ and starting the photoreaction. We conclude that time-resolved photoaffinity labeling can convalently mark protein structures involved in receptor functions. Of special interest is the observation that antagonists also induce a conformational change in the receptor protein.

Acetylcholine↗
PubMed1985

A neutron diffraction study on the location of the polyene chain of retinal in bacteriorhodopsin.

We report on the location of the chain part of the retinylidene chromophore in the projected density of bacteriorhodopsin as determined by neutron diffraction from the two-dimensional purple membrane lattice. For this purpose, partially deuterated retinal was synthesized containing 10 deuterons at positions C-8, C-10, C-12, C-14, C-19(3), and C-20(3) of the polyene chain. Two sets of dark-adapted samples were prepared in entirely different ways: (i) Deuterated retinal was incorporated biosynthetically during growth of the bacteria by using the mutant JW5, which is deficient in the synthesis of retinal. (ii) The chromophore was converted to retinal oxime, the resulting colorless apomembrane was regenerated with deuterated retinal, and the residual retinal oxime was removed by washing with bovine serum albumin. Characterization of these samples by x-ray diffraction, absorption, and flash spectroscopy showed that they were identical to native purple membrane samples as judged by these criteria. Fourier difference maps were calculated from the differences in inplane diffraction from the deuterated membranes and from protonated samples that were prepared in exactly the same way. At 8.7 A resolution, both maps show a single major peak at the same position with the center of mass of the labeled part of the chain (C-11) between helices 6 and 3 but closer to helix 6. It appears likely that the COOH-terminal helix G, to which retinal is attached at lysine-216, is either helix 2 or 6.

Apoproteins↗
PubMed1983

Photochemical cycle and light-dark adaptation of monomeric and aggregated bacteriorhodopsin in various lipid environments.

Spectral changes of bacteriorhodopsin (BR) reflecting its photochemical cycle and light-dark adaptation were monitored in order to study the effect of protein-protein and protein-lipid interactions on these reactions. For this purpose, the light-driven proton pump BR was reconstituted with various lipids, i.e., dimyristoyl- and dipalmitoyl-phosphatidylcholine, soybean phospholipids, and diphytanoyllecithin. In these vesicle systems, BR is monomeric above the lipid phase transition and above molar lipid to BR ratios of about 80. Well below the phase transition, BR is aggregated in a hexagonal lattice as in the purple membrane. This allows, on the one hand, comparison of monomeric and aggregated BR in the respective vesicle systems and, on the other hand, comparison of reconstituted BR with BR in the native purple membrane. The photoreaction cycle of all-trans-BR accompanying proton translocation proceeds via the same intermediates in the monomeric and aggregated pigment. Furthermore, both the rate and the activation energy for the decay of the cycle intermediate M-410 are independent of the aggregation state. From the results, we conclude that the functional unit responsible for BR's photocycle is the monomer itself. This is in accordance with previous observations that BR monomers are able to translocate protons during illumination [Drencher, N. A., & Heyn, M.P. (1979) FEBS Lett. 108, 307-310]. The light-dark adaptation reaction, however, is affected by BR's aggregation state. In the case of the monomer, the extent of light adaptation, i.e., the fraction of BR molecules containing 13-cis-retinal as chromophore which is converted by illumination to the respective pigment with the all-trans isomer, is reduced by 50% or more, and the rate of dark adaptation is slowed down about 2.5 times. For these properties too, the monomer is functional, but with a reduced efficiency. This indicates regulatory control by neighboring BR molecules. The rate of the photocycle as well as of dark adaptation is strongly affected by the chemical nature of the lipids used for reconstitution but not by the physical state of the lipid phase.

Bacteriorhodopsins↗
PubMed1983

Fluorescence energy transfer from diphenylhexatriene to bacteriorhodopsin in lipid vesicles.

Fluorescence energy transfer between the donor diphenylhexatriene (DPH) and the acceptor retinal and fluorescence depolarization of DPH are used to test current theories for fluorescence energy transfer in two-dimensional systems and to obtain information on the effect of the intrinsic membrane protein, bacteriorhodopsin, on the order and dynamics of the lipid phase. Increasing the surface concentration of acceptors by raising the protein to lipid ratio leads to a decrease in the mean fluorescence lifetime by up to a factor of four. When the acceptor concentration is reduced at a fixed protein to lipid ratio by photochemical destruction of retinal, the lifetime increases and reaches approximately the value observed in protein-free vesicles when the bleaching is complete. The shape of the decay curve and the dependency of the mean lifetime on the surface concentration of acceptors are in agreement with theoretical predictions for a two-dimensional random distribution of donors and acceptors. From this analysis a distance of closest approach between donors and acceptors of approximately 18 A is obtained, which is close to the effective radius of bacteriorhodopsin (17 A) and consistent with current ideas about the location of retinal in the interior of the protein. In the absence of energy transfer (bleached vesicles), the steady-state fluorescence anisotropy, -r, of DPH is considerably lower than in the corresponding unbleached vesicles, indicating that the effect of energy transfer must be taken into account when interpreting -r in terms of order and dynamics.

Bacteriorhodopsins↗
PubMed1981

Calorimetric and fluorescence depolarization studies on the lipid phase transition of bacteriorhodopsin--dimyristoylphosphatidylcholine vesicles.

The thermotropic lipid phase transition of dimyristoylphosphatidylcholine vesicles reconstituted with bacteriorhodopsin was investigated as a function of the lipid to protein ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. Two attractive features of this system are that the lipid phase transition induces lipid-protein segregation and that the state of aggregation of the protein is known. Above the lipid phase transition and above molar lipid to protein ratios of about 100, bacteriorhodopsin is monomeric. Well below the phase transition, bacteriorhodopsin is aggregated in a hexagonal protein lattice. With increasing amounts of incorporated bacteriorhodopsin, the calorimetric transition broadens, and a second component develops at a temperature which is lower than that of the unperturbed transition. The latter transition was assigned to the disaggregation of the bacteriorhodpsin lattice which occurs 6-7 degrees C below the phase transition of the protein-free lipids according to previous measurements. The van't Hoff enthalpy of the phase transition, as determined from the fluorescence depolarization of diphenylhexatriene, is in surprisingly good agreement with that obtained from differential scanning calorimetry over a wide range of lipid to protein ratios. The differential scanning calorimetry data can be simulated on the basis of a model which takes the protein segregation and crystallization specifically into account. The essential feature of this model is that calorimetrically detectable lipid melting occurs in the temperature region of the protein crystallization, since for the disaggregation of the close packed bacteriorhodopsin lattice additional lipids of an intermediate chain conformation are required.

Bacteriorhodopsins↗
PubMed1981

Lipid--protein interactions in bacteriorhodopsin--dimyristoylphosphatidylcholine vesicles.

Bacteriorhodopsin (BR) was incorporated into large unilamellar dimyristoyl- and dipalmitoylphosphatidylcholine vesicles (100--300-nm radius). The effect of this intrinsic membrane protein on the order and dynamics of the lipid and on the cooperativity and transition temperature (Tc) of the gel to liquid-crystalline phase transition was investigated as a function of the lipid:protein ratio (L/BR). The lipid phase transition induces protein segregation. Above Tc, bacteriorhodopsin is in the monomeric state. Below Tc, BR is aggregated in the same hexagonal lattice as in the purple membrane (PM). In this reconstituted system, BR has a photochemical cycle similar to that in the PM and is active as a light-driven proton pump. The lipid phase transition which was monitored by using the steady-state anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) broadens with decreasing L/BR but occurs at approximately the same Tc. Below Tc, the fluorescence anisotropy of DPH is quite high (0.35) and independent of the L/BR. Above Tc, however, the anisotrophy increases markedly with decreasing L/BR. It was recently pointed out that the fluorescence anisotropy of probes like DPH contains information not only on the dynamics (correlation times) but also on the order parameters of the lipids [Heyn, M. P. (1979) FEBS Lett. 108, 359--364]. The most likely explanation of the observed increase in anisotropy above Tc is that the perturbation of the lipid bilayer caused by the incorporation of BR leads both to an increase in order and to a slowing of the rotational diffusion of the lipids (increased viscosity). In agreement with this latter dynamical effect, the rotational diffusion constant of BR itself decreases above Tc with decreasing L/BR. Above Tc, the membrane viscosity as determined from the rotational diffusion constant of BR is at least 1.5 times larger than that obtained from the fluorescence depolarization of DPH. The formation of the BR lattice as a function of temperature was followed by using the circular dichroism (CD) exciton effect together with measurements of the rotational diffusion of BR. Both methods show similar transition curves for the protein crystallization whose midpoints, however, occur several degrees below Tc.

Bacteriorhodopsins↗
PubMed1981

Transmembranous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers.

Various methods to incorporate bacteriorhodopsin in black lipid membranes are reported. Both purple membrane patches and monomeric bacteriorhodopsin were used as starting material. The incorporation of bacteriorhodopsin into planar lipid bilayers was achieved by the following methods. (i) Purple membrane patches were transferred from water to solutions of lipids in n-alkanes. Black membranes were formed from such organic suspensions. (ii) Lipid layers containing solvent and purple membranes were spread on an air/water interface. These layers were used to form planar bilayers. (iii) Vesicles containing purple membranes or monomeric bacteriorhodopsin were spread on an air/water interface and, from the resulting layer, bilayers were formed. On illumination, steady-state photocurrents were observed in all three cases, indicating that these methods lead to functional transmembranous integration of the protein in the planar black lipid membrane. The influence of an applied electric field on the pumping process was studied on membranes formed by using method i. At approximately 200 mV, the photocurrent tends to zero. Furthermore, it was possible to make planar lipid bilayers photoelectrically active by adding vesicles containing monomeric bacteriorhodopsin to the bathing solution. Because, in this case, only transient photocurrents were observed, it can be concluded that the vesicles are attached to but not fused with the black lipid membrane.

Bacteriorhodopsins↗
PubMed1980

Lateral segregation of proteins induced by cholesterol in bacteriorhodopsin-phospholipid vesicles.

Bacteriorhodopsin in dimyristoylphosphatidylcholine vesicles is randomly distributed in the plane of the membrane and exhibits rotational diffusion above the gel to liquid-crystalline phase transition. Incorporation of cholesterol results in loss of rotational mobility of bacteriorhodopsin, which on the basis of electron microscopy and CD measurements can be assigned to the formation of protein aggregates. It is concluded that bacteriorhodopsin is soluble in the fluid phosphatidylcholine phase but segregates when cholesterol is present in the lipid bilayer.

Bacteriorhodopsins↗