Biomedical subjects

M Sheves

Publications and source records attributed to M Sheves.

At least 37 records · Page 2Linked to original sources

PubMed1997

Microsecond atomic force sensing of protein conformational dynamics: implications for the primary light-induced events in bacteriorhodopsin.

In this paper a new atomic force sensing technique is presented for dynamically probing conformational changes in proteins. The method is applied to the light-induced changes in the membrane-bound proton pump bacteriorhodopsin (bR). The microsecond time-resolution of the method, as presently implemented, covers many of the intermediates of the bR photocycle which is well characterized by spectroscopical methods. In addition to the native pigment, we have studied bR proteins substituted with chemically modified retinal chromophores. These synthetic chromophores were designed to restrict their ability to isomerize, while maintaining the basic characteristic of a large light-induced charge redistribution in the vertically excited Franck-Condon state. An analysis of the atomic force sensing signals lead us to conclude that protein conformational changes in bR can be initiated as a result of a light-triggered redistribution of electronic charge in the retinal chromophore, even when isomerization cannot take place. Although the coupling mechanism of such changes to the light-induced proton pump is still not established, our data question the current working hypothesis which attributes all primary events in retinal proteins to an initial trans<==>cis isomerization.

Bacteriorhodopsins↗

PubMed1997

Reducing the flexibility of retinal restores a wild-type-like photocycle in bacteriorhodopsin mutants defective in protein-retinal coupling.

The thermal re-isomerization of retinal from the 13-cis to the all-trans state is a key step in the final stages of the photocycle of the light-driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu-93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10-C11 (9,11-bridged retinal) or C12-C13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 --> Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein-retinal coupling in the bacteriorhodopsin photocycle.

Alanine↗

PubMed1997

Trp86 --> Phe replacement in bacteriorhodopsin affects a water molecule near Asp85 and light adaptation.

Illumination of the Trp86 --> Phe mutant of bacteriorhodopsin causes anomalous light adaptation, i.e., isomerization of the retinal from all-trans to 13-cis, 15-syn. FTIR spectral analysis shows that illumination at 250 K yields two 13-cis photoproducts, the conventional 13-cis, 15-syn state, BR(C), and another termed BR(X). BR(X) is different from BR(C) because it has a lower N-H in-plane bending frequency and a higher C14-C15 stretching frequency, as well as an absence of coupling between these modes. BR(X), which is stable at 275 K, is more abundant in the photosteady state produced by longer wavelength light and detected as the only photoproduct at 170 K. Its different structural features result from distortion of the C14-C15 bond of the chromophore. In the W86F mutant protein, the small structural changes of a water molecule in the conversion between the all-trans and 13-cis, 15-syn forms and in the formation of the K photointermediate are absent, but the larger changes of water molecule(s) that normally occur in the L and M intermediates are present. We propose that Trp86, together with Asp85, is involved in binding the water molecule and in preventing the formation of the 13-cis, 15-syn photoproducts, BR(C) and BR(X), when the wild type protein is illuminated.

Aspartic Acid↗

PubMed1997

Interaction between Asp-85 and the proton-releasing group in bacteriorhodopsin. A study of an O-like photocycle intermediate.

Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted O1. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these O1 species was investigated by studying a series of effects, primarily their red light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid-base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The O1 species of pigments I and II (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in O1, Asp-85 is protonated, a process associated with proton uptake from the extracellular side. Subsequent proton release (to the same side of the membrane) occurs from Glu-204 (or from a group closely interacting with it) prior to the decay of O1. At high pH (>9), O1 reversibly converts to a purple form, due to deprotonation of Asp-85, while at still higher pH (> 11), a blue-shifted species characterized by a deprotonated Schiff base is generated. These transitions constitute the first demonstration of the titration of a photocycle intermediate of a retinal protein. The respective pKa values are determined and discussed in relation to those pertaining to the unphotolyzed (dark-adapted) pigments. It appears that the pKa values are controlled by a hydrogen bond network involving water molecules, which binds the protonated Schiff base with Asp-85 and Glu-204. The disruption of this network in pigments I-III may also be responsible for the long lifetime of the O1 species, due to the inhibition of thermal trans-13-cis isomerization. The results are relevant to the molecular mechanism of the photocycles of both 13-cis- and all-trans-bR, primarily to the nature and to the deprotonation mechanism of the proton-releasing group.

Arylsulfonates↗

PubMed1997

Atomic Force Sensing of Light-Induced Protein Dynamics with Microsecond Time Resolution in Bacteriorhodopsin and Photosynthetic Reaction Centers

This paper reports on experiments that have monitored protein microsecond dynamics with a cantilevered near-field optical glass fiber. In these experiments two photoactive proteins, bacteriorhodopsin (bR) and the photosynthetic reaction center (PS I), are used to demonstrate that such probes can measure light-induced microsecond protein dynamics even though the resonance frequencies of the glass cantilevers used are on the order of a few hundred kilohertz. In the case of the light-driven proton pump, bR, the light-induced atomic force sensing (AFS) signal is negative (indicating contraction) in the microsecond time domain of the L photointermediate and becomes positive (corresponding to expansion) in the subsequent M intermediate that lives for milliseconds. Double pulse experiments from M to bR show that the latter process reverses the AFS signal. Thus, the AFS structural changes are coupled with the (optical) photocycle intermediates. Light-induced contraction and expansion phenomena are also observed in the case of PS I. In both systems the time regime of the dynamic phenomena that have been measured with AFS is five orders of magnitude faster than the fastest previously recorded atomic force detection of dynamic phenomena. This advance portends a new era in dynamic imaging of protein conformational changes.

Journal Article↗

PubMed1997

Evidence for a controlling role of water in producing the native bacteriorhodopsin structure.

The experiments reported in this paper, based on reconstitution of bacteriorhodopsin (bR) from apomembrane at varying environmental conditions, demonstrate that the presence of water is a controlling factor in generating a native wild-type bR conformation. If water is lacking during this reconstitution process, then a non-native bR structure is formed that exhibits altered M formation and decay kinetics, as well as different behavior following extensive dehydration. It is shown that mutants affecting the ability of bR to form appropriate structures of water in specific protein cavities also affect the ability to generate a native bR conformation. The results suggest that aspartic acid 96 plays a major role in anchoring the appropriate water structure conformation associated with bR. It is also demonstrated that the glutamic acid 204 residue is pivotal in controlling the protein/water affinity. This water affinity can be further controlled by modifying the charge environment of the protein with altered pH. These data, based on kinetic absorption spectroscopy and Fourier transform infrared spectroscopy, highlight the central role of water in this protein.

Aspartic Acid↗

PubMed1996

Steric interaction between the 9-methyl group of the retinal and tryptophan 182 controls 13-cis to all-trans reisomerization and proton uptake in the bacteriorhodopsin photocycle.

The hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds; no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pKa of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant the first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in providing tight coupling between chromophore isomerization and the late proton transfer steps.

Halobacterium↗

PubMed1996

Evidence that aspartate-85 has a higher pK(a) in all-trans than in 13-cisbacteriorhodopsin.

Three experimental observations indicate that the pK(a) of the purple-to-blue transition (the pK(a) of Asp-85) is higher for all-trans-bR(1) than for 13-cis-bR. First, light adaptation of bacteriorhodopsin (bR) at pHs near the pK(a) of Asp-85 causes an increase in the fraction of the blue membrane present. This transformation is reversible in the dark. Second, the pK(a) of the purple-to-blue transition in the dark is lower than that in the light-adapted bR (pK(a)(DA) = 3.5, pK(a)(LA) = 3.8 in 10 microM K(2)SO(4)). Third, the equilibrium fractions of 13-cis and all-trans isomers are pH dependent; the fraction of all-trans-bR increases upon formation of the blue membrane. Based on the conclusion that thermal all-trans <=> 13-cis isomerization occurs in the blue membrane rather than in the purple, we have developed a simple model that accounts for all three observations. From the fit of experimental data we estimate that the pK(a) of Asp-85 in 13-cis-bR is 0.5 +/- 0.1 pK(a) unit less than the pK(a) of all-trans-bR. Thus in 10 microM K(2)SO(4), pK(a)(c) = 3.3, whereas pK(a)(t) = 3.8.

Aspartic Acid↗

PubMed1996

Directly probing rapid membrane protein dynamics with an atomic force microscope: a study of light-induced conformational alterations in bacteriorhodopsin.

This paper demonstrates that an atomic force microscope can be used to directly monitor rapid membrane protein dynamics. For this demonstration the membrane-bound proton pump, bacteriorhodopsin, has been investigated. It has been unequivocally shown that the light-induced dynamic alterations that have been observed do not arise from external artifacts such as heating of the sample by the incident light, but that these changes can be directly linked to the light-induced protein conformational alterations in this membrane. In essence, it has been shown that the light energy absorbed by bacteriorhodopsin is converted not only to chemical energy but also to mechanical energy. In summary a new ultrasensitive tool is described for monitoring the molecular dynamics of materials with wide applicability to fundamental and applied science.

Bacteriorhodopsins↗

PubMed1995

Influence of the 9-methyl group of the retinal on the photocycle of bacteriorhodopsin studied by time-resolved rapid-scan and static low-temperature Fourier transform infrared difference spectroscopy.

The photocycle of bacteriorhodopsin (BR) regenerated with all-trans-9-demethylretinal was investigated by time-resolved rapid-scan Fourier transform infrared difference spectroscopy, by static low-temperature difference spectroscopy at 80, 170, and 213 K and by static steady-state difference spectroscopy at 278 K. In addition, the formation and decay of M intermediate was monitored at 412 nm with conventional flash photolysis experiments. Our data show that the removal of the 9-methyl group strongly changes the photocycle of BR. The reaction cycle is slowed down about 250-fold. The photoreaction is characterized by a slow rise of the M intermediate and by a very long-lived N intermediate. No O intermediate could be observed. The low-temperature spectra indicate that already at 80 K a KL-like photoproduct is formed. L can be obtained as in native BR at 170 K, but its decay appears to be inhibited, since it can still be observed at 213 K and high pH, in addition to the M intermediate. As in native BR, the 15-hydrogen out-of-plane modes of the L and N intermediates (observed in 2H2O) are very similar. Evidence for the existence of three N substates which differ in the protonation state of Asp96 and in the amide I bands is presented. This is explained by the extremely slowed-down reisomerization of the chromophore. The results are discussed with respect to alterations in the chromophore-protein interaction, caused by the removal of the 9-methyl group.

Bacteriorhodopsins↗

PubMed1995

The complex extracellular domain regulates the deprotonation and reprotonation of the retinal Schiff base during the bacteriorhodopsin photocycle.

During the L-->M reaction of the bacteriorhodopsin photocycle the proton of the retinal Schiff base is transferred to the anionic D85. This step, together with the subsequent reprotonation of the Schiff base from D96 in the M-->N reaction, results in the translocation of a proton across the membrane. The first of these critical proton transfers occurs in an extended hydrogen-bonded complex containing two negatively charged residues (D85 and D212), two positively charged groups (the Schiff base and R82), and coordinated water. We simplified this region by replacing D212 and R82 with neutral residues, leaving only the proton donor and acceptor as charged groups. The D212N/R82Q mutant shows essentially normal proton transport, but in the photocycle neither of this protein nor of the D212N/R82Q/D96N triple mutant does a deprotonated Schiff base (the M intermediate) accumulate. Instead, the photocycle contains only the K, L, and N intermediates. Infrared difference spectra of D212N/R82Q and D212N/R82Q/D96N demonstrate that although D96 becomes deprotonated in N, D85 remains unprotonated. On the other hand, M is produced at pH > 8, where according to independent evidence the L<==>M equilibrium should shift toward M. Likewise, M is restored in the photocycle when the retinal is replaced with the 14-fluoro analogue that lowers the pKa of the protonated Schiff base, and now D85 becomes protonated as in the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗

PubMed1995

pKa of the protonated Schiff base and aspartic 85 in the bacteriorhodopsin binding site is controlled by a specific geometry between the two residues.

The structure and function of the light-driven proton pump bacteriorhodopsin appear to be determined by the exact geometrical conformation of specific groups in the retinal binding site, including bound water molecules. This applies to the pKa values of the protonated Schiff base, which links the retinal chromophore to Lys216, and to Asp85. In the present work we show that the geometrical constraints imposed by the ring structures of several synthetic retinals can induce substantial changes in the pKa values of the Schiff base and of Asp85. Thus, the artificial pigments derived from 13-demethyl-11,14-epoxyretinal (2) and 13-demethyl-9,12-epoxyretinal (3) show protonated Schiff base pKa values of 8.2 +/- 0.1 and 9.1 +/- 0.1, respectively, as compared with 13.3 in the native (all-trans-retinal) pigment. We also suggest that in both systems the pKa of Asp85 increases from 3.2 in the native bR to above 9. Analogous, though smaller, effects are obtained for artificial bR pigments derived from 12,14-ethanoretinal (4), 11,13-propanoretinal (5), 11,13-ethanoretinal (6), and p-(CH3)2N-C6H4-HC = CH-C(CH3) = CH-CHO 7. The effects of geometry on the pKa values (those on Asp85 being more pronounced) are attributed to the disruption of the original, well-defined, structure in which the Schiff base and its Asp85 counterion are bridged by bound water molecules. These results are the first to show that it is possible to modify the pKa values of the Schiff base and Asp85 in appropriate artificial pigments, without inducing intrinsic pKa changes in the chromophore or introducing a mutation in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗

PubMed1995

Time-resolved titrations of the Schiff base and of the Asp85 residue in artificial bacteriorhodopsins.

Deprotonation/protonation processes involving the retinal Schiff base and the Asp85 residue play dominant roles in the light-induced proton pump of bacteriorhodopsin (bR). Although the pKa values of these two moieties in unphotolyzed bR are well established, the kinetics of the respective titrations in the native pigment are difficult to interpret, primarily due to the extreme (nonphysiological) pKa values of the two moieties (12.2 +/- 0.2 and 2.7, in 0.1 M NaCl, for the Schiff base and for Asp85, respectively). These difficulties are circumvented by applying stopped-flow techniques, time resolving the titrations of several artificial bRs in which the pKa values of the above two residues are substantially modified: 13-CF3 bR, pKa (Schiff base) = 8.2 +/- 0.2; 13-demethyl-11,14-epoxy bR, pKa (Schiff base) = 8.2 +/- 0.1 (in 0.1 M NaCl); aromatic bR, pKa (Asp85) = 5.2 +/- 0.1 (in water). The R82Q bR mutant, pKa (Asp85) congruent to 7.2 was also employed. A major objective was to verify whether the basic relationships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium constant as the ratio of the forward and back rate constants) are also obeyed by the Schiff base and Asp85 moieties. We found that this is the case for the Schiff base in the pH range between 7 and 9 but not at lower pH. These observations led to the conclusion that the Schiff base is titrable from the outside medium via a proton channel, which becomes saturated, and thus rate determining, below pH approximately equal to 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗

PubMed1995

The role of water in retinal complexation to bacterio-opsin.

A system is described that allows for the delineation of the factors that effect the complexation of retinal to the apoprotein of bacteriorhodopsin. This complexation is investigated in various states of hydration, in H2O and D2O, at a variety of pH levels, with mutant membranes and labeled retinals. The complexation reaction was also investigated using absorption spectroscopy and vibrational spectra using difference Fourier transform infrared spectroscopy. The results demonstrate the crucial role of water in controlling the protein conformations that lead to protein/ligand binding reactions and begin to shed new light on the protein control of a reaction that normally cannot take place in an aqueous medium.

Bacteriorhodopsins↗

PubMed1995

Chloride effect on the early photolysis intermediates of a gecko cone-type visual pigment.

Nanosecond laser photolysis measurements were conducted on the cone-type visual pigment P521 in digitonin extracts of Tokay gecko (Gekko gekko) retina containing physiological chloride ion levels and also on samples which had been chloride depleted or which contained high levels (4 M) of chloride. Absorbance difference spectra were recorded at a sequence of time delays from 30 ns to 60 microseconds following excitation with a pulse of either 532- or 477-nm actinic light. Global analysis showed the kinetic decay data for gecko pigment P521 to be best fit by two exponential processes under all chloride conditions. The initial photoproduct detected had a broad spectrum characteristic of an equilibrated mixture of a Batho P521 intermediate with its blue-shifted intermediate (BSI P521) decay product. The first exponential process was assigned to the decay of this mixture to the Lumi P521 intermediate. The second exponential process was identified as the decay of Lumi P521 to Meta I P521. The initial photoproduct's spectrum exhibited a strong dependence on chloride concentration, indicating that chloride affects the composition of the equilibrated mixture of Batho P521 and BSI P521. These results suggest that the affinity for chloride is reduced approximately 5-fold in the Batho P521 intermediate and approximately 50-fold in the BSI P521 intermediate. Chloride concentration also affects the apparent decay rate of the equilibrated mixture. When the apparent decay rate is corrected for the composition of the equilibrated mixture, a relatively invariant microscopic rate constant is obtained for BSI decay (k = 1/55 ns-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effects of substitution of tyrosine 57 with asparagine and phenylalanine on the properties of bacteriorhodopsin.

Tyrosine 57 is one of the residues present in the retinal binding pocket and is conserved in all the halophilic retinal proteins. We have studied mutants of bacteriorhodopsin, expressed in Halobacterium salinarium, in which tyrosine 57 is replaced by an asparagine (Y57N) or phenylalanine (Y57F). In Y57N the photocycle proceeds only up to the L intermediate; no M is formed at neutral pH. The lifetime of L intermediate is extremely long, ca. 500 ms. Proton release is severely affected in both the mutants which suggests that Y57 is associated with the proton release pathway. By comparing the pH-induced absorption changes in the UV in Y57N and Y57F with those in the wild-type (WT), we determined that the pKa of Y57 is 10.2. In Y57F, which shows M formation, the rate constant of the L-->M transition is pH dependent (pKa 8.7) suggesting that Y57 is probably not the residue that normally controls the transition into the alkaline photocycle. Y57 is either part of the counterion complex or in close proximity to D85 since its mutation influences the pKa of Asp85. In Y57F the pKa of D85 is approximately 4.9 (compared to approximately 2.9 in the WT). The Y57N mutant shows two pKa's in the purple to blue transition, approximately 3.8 and < 1. In the presence of hydroxylamine, at neutral pH, Y57N is stable in the dark but bleaches very rapidly upon illumination compared to the WT. Since the lifetime of L intermediate is long in Y57N, we suggest that the Schiff base becomes accessible to hydroxylamine in this state.

Asparagine↗

PubMed1995

Primary picosecond molecular events in the photoreaction of the BR5.12 artificial bacteriorhodopsin pigment.

The picosecond dynamics of the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a retinal in which a five-membered ring spans the C-12 to C-14 positions of the polyene chain (BR5.12) is examined by using time-resolved absorption and fluorescence and resonance Raman spectroscopy. The ring within the retinal chromophore of BR5.12 blocks the C-13 = C-14 isomerization proposed to be a primary step in the energy storage/transduction mechanism in the BR photocycle. Relative to the native BR pigment (BR-570), the absorption spectrum of BR5.12 is red-shifted by 8 nm. The fluorescence spectrum of BR5.12 closely resembles that of BR-570 although the relative fluorescence yield is higher (approximately 10-fold). Picosecond transient absorption (4-ps pulses, 568-662 nm) measurements reveal an intermediate absorbing to the red side of BR5.12. Kinetic fits show that the red-absorbing intermediate appears within < 3 ps and decays with a time constant of 17 +/- 1 ps to form only BR5.12. No emission in the 650- to 900-nm region can be attributed to the red-absorbing species. Since rotation around C-12 - C-13 and isomerization around C-13 = C-14 are prevented in BR5.12, these results demonstrate that motion in these regions of the retinal is (i) necessary to form the K-like intermediate observed in the native BR-570 photocycle and (ii) not necessary to form a red-absorbing intermediate that has spectral and kinetic properties analogous to those of J-625 in the native BR photocycle. Discussions of the excited and ground electronic state assignments for the intermediate observed in the BR5.12 photoreaction are presented.

Bacteriorhodopsins↗

PubMed1995

Molecular dynamics study of the M412 intermediate of bacteriorhodopsin.

Molecular dynamics simulations have been carried out to study the M412 intermediate of bacteriorhodopsin's (bR) photocycle. The simulations start from two simulated structures for the L550 intermediate of the photocycle, one involving a 13-cis retinal with strong torsions, the other a 13,14-dicis retinal, from which the M412 intermediate is initiated through proton transfer to Asp-85. The simulations are based on a refined structure of bR568 obtained through all-atom molecular dynamics simulations and placement of 16 waters inside the protein. The structures of the L550 intermediates were obtained through simulated photoisomerization and subsequent molecular dynamics, and simulated annealing. Our simulations reveal that the M412 intermediate actually comprises a series of conformations involving 1) a motion of retinal; 2) protein conformational changes; and 3) diffusion and reconfiguration of water in the space between the retinal Schiff base nitrogen and the Asp-96 side group. (1) turns the retinal Schiff base nitrogen from an early orientation toward Asp-85 to a late orientation toward Asp-96; (2) disconnects the hydrogen bond network between retinal and Asp-85 and tilts the helix F of bR, enlarging bR's cytoplasmic channel; (3) adds two water molecules to the three water molecules existing in the cytoplasmic channel at the bR568 stage and forms a proton conduction pathway. The conformational change (2) of the protein involves a 60 degrees bent of the cytoplasmic side of helix F and is induced through a break of a hydrogen bond between Tyr-185 and a water-side group complex in the counterion region.

Amino Acid Sequence↗