Biomedical subjects

R Needleman

Publications and source records attributed to R Needleman.

At least 55 records · Page 3Linked to original sources

PubMed1996

Conformation and dynamics of [3-13C]Ala- labeled bacteriorhodopsin and bacterioopsin, induced by interaction with retinal and its analogs, as studied by 13C nuclear magnetic resonance.

13C nuclear magnetic resonance (NMR) spectra of [3-13C]Ala-labeled bacteriorhodopsin (bR), bacterioopsin (bO), and regenerated bR with retinal or bO complex with retinal analogs were recorded in order to gain insights into how the conformation and dynamics of apoprotein (bO) vary with or without retinal or its analogs. First, we assigned the 13C NMR peak resonating at 16.3 ppm to Ala 53 of both bR and bO, which appears to contact the side chain of Lys 216 at the site of the Schiff base in the former, utilizing the 13C NMR peaks of A53V and A53G proteins in comparison with those of wild-type bR and bO. Characteristic spectral differences between the apoprotein and bR were observed upon removal of the retinal: the changes of the peak intensities at 16.4, 15.9, and 16.9 ppm are notable. We found that the loops (17.4 ppm) and transmembrane alpha II helical region (15.9 ppm) acquired motional freedom with a correlation time of 10(-5)s when the retinal was removed, as detected by proton spin-lattice relaxation times in the rotating frame. A 13C NMR spectrum very similar to that of native bR was recorded when bR was regenerated by addition of retinal to bO. On the other hand, the addition of the retinal analogs retinol or beta-ionone, which are bound in the retinal binding site but are incapable of forming a Schiff base to the apoprotein, caused distinct spectral changes different from those of bR, as manifested from the displacements of 13C chemical shifts. These spectral changes must be ascribed to significant conformational changes of apoprotein at various locations in the protein, including the site of Ala 53 induced by modified interaction between the apoprotein and chromophore.

Alanine↗

PubMed1996

Proton transport by halorhodopsin.

In halorhodopsin from Natronobacterium pharaonis, a light-driven chloride pump, the chloride binding site also binds azide. When azide is bound at this location the retinal Schiff base transiently deprotonates after photoexcitation with light > 530 nm, like in the light-driven proton pump bacteriorhodopsin. As in the photocycle of bacteriorhodopsin, pyranine detects the release of protons to the bulk. The subsequent reprotonation of the Schiff base is also dependent on azide, but with different kinetics that suggest a shuttling of protons from the surface as described earlier for halorhodopsin from Halobacterium salinarium. This azide-dependent, bacteriorhodopsin-like photocycle results in active electrogenic proton transport in the cytoplasmic to extracellular direction, detected in cell envelope vesicle suspensions both with a potential-sensitive electrode and by measuring light-dependent pH change. We conclude that in halorhodopsin an azide bound to the extracellular side of the Schiff base, and another azide shuttling between the Schiff base and the cytoplasmic surface, fulfill the functions of Asp-85 and Asp-96, respectively, in bacteriorhodopsin. Thus, although halorhodopsin is normally a chloride ion pump, it evidently contains all structural requirements, except an internal proton acceptor and a donor, of a proton pump. This observation complements our earlier finding that when a chloride binding site was created in bacteriorhodopsin through replacement of Asp-85 with a threonine, that protein became a chloride ion pump.

Azides↗

PubMed1996

Effects of arginine-82 on the interactions of internal water molecules in bacteriorhodopsin.

Arg82, one of the residues near the protonated Schiff base of bacteriorhodopsin, facilitates proton release to the medium during the L-to-M reaction of the photocycle, but retards the rate of proton transfer from the Schiff base to Asp85. In order to understand the role of Arg82 in these processes, the structural changes upon formation of the M intermediate were studied by Fourier transform infrared spectroscopy of the hydrated films of Arg82 mutants at pH 9.5. The negative band at 1700 cm-1 in the BR --> M spectrum due to the deprotonation of Glu204 was absent when Arg82 was replaced with alanine (R82A), but present with small amplitude when residue 82 was a glutamine (R82Q), or a lysine (R82K), with a shift to 1696 cm-1. The O-H stretch of water at 3643 cm-1 is shifted toward a lower frequency in R82Q, R82K, and R82A in the unphotolyzed state. However, R82Q retains a fraction of the unshifted band. Another O-H stretch is prominent in R82Q around 3625 cm-1 but absent in R82A and probably in R82K. In parallel, R82Q retains a fraction of the slow component of the formation of the M intermediate, which is almost completely absent in R82K and R82A. These results, along with previous data for the mutants of Glu204, suggest that the guanidium group of Arg82 influences the H-bonding of water molecules located close to Asp85 and Arg82-Glu204 regions, and the rate of proton transfer from the Schiff base to Asp85. The amide group of Gln82 can substitute for it but weakly.

Arginine↗

PubMed1996

Hydrogen bonds of water and C==O groups coordinate long-range structural changes in the L photointermediate of bacteriorhodopsin.

Fourier transform infrared spectra of light-adapted bacteriorhodopsin exhibit a band at 1618 cm(-1) that shifts to 1625 cm(-1) upon formation of the L intermediate. It is assigned to the peptide C==O of Val49 from the fact that it shifts in [1-(13)C]valine-labeled bacteriorhodopsin and appears perturbed in the Val49-->Met mutant. The intensity of the BR-->L difference band is reduced in the Thr46-->Val mutant but restored by the additional mutation of Asp96-->Asn. These intensity changes are closely correlated with the H-bonding change of water olecules, suggesting that the peptide C==O of Val49 is hydrated. This could arise in the Thr46-->Val mutant because of perturbation of the C==O of Val46, which points toward Val49. The Val49-->Ala mutation influences a peptide N-H, presumably of Val49, and the carboxylic C==O of Asp96, as well as water molecules proximal to Asp85. Conversely, the water molecule assumed to be in the cavity that arises from the missing two methyl groups in V49A could be affected in the mutant of Asp96-->Asn. We propose that the perturbation exerted on Asp85 by the Schiff base in the L intermediate is transmitted to Asp96 through H-bonding of water molecules in the Asp85-Val49 region, the C==O of Val49, H-bonding between Val49 and Thr46, and H-bonding between Thr46 and Asp96.

Amino Acid Sequence↗

PubMed1996

A linkage of the pKa's of asp-85 and glu-204 forms part of the reprotonation switch of bacteriorhodopsin.

Because asp-85 is the acceptor of the retinal Schiff base proton during light-driven proton transport by bacteriorhodopsin, modulation of its pKa in the photocycle is to be expected. The complex titration of asp-85 in the unphotolyzed protein was suggested [Balashov, S. P., Govindjee, R., Imasheva, E. S., Misra, S., Ebrey, T. G., Feng, Y., Crouch, R. K., & Menick, D. R (1995) Biochemistry 34, 8820-8834] to reflect the dependence of this residue on the protonation state of another, unidentified group. From the pH dependencies of the rate constant for the thermal equilibration of retinal isomeric states (dark adaptation) and the deprotonation kinetics of the Schiff base during the photocycle in the E204Q and E204D mutants, we identify the residue as glu-204. The nature of its interaction with asp-85 is that at neutral pH either residue can be anionic but not both. This is consistent with our recent finding that glu-204 is the origin of the proton released to the extracellular surface upon protonation of asp-85 during the transport. We propose, therefore, that the following series of events occur in the photocycle. Protonation of asp-85 in the proton equilibrium with the Schiff base of the photoisomerized retinal results in the dissociation of glu-204 and proton release to the extracellular surface. The deprotonation of glu-204, in turn, raises the pK(a) of asp-85, and the equilibrium with the Schiff base shifts toward complete proton transfer. This constitutes the first phase of the reprotonation switch because it excludes asp-85 as a donor in the reprotonation of the Schiff base that follows. The sequential structural changes of the protein that ensue, detected earlier by diffraction, are suggested to facilitate the change of the access of the Schiff base toward the cytoplasmic side as the second phase of the switch, and the lowering the pKa of asp-96, so as to make it a proton donor, as the third phase.

Amino Acid Sequence↗

PubMed1996

Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction.

X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5 degrees Celsius. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.

Aspartic Acid↗

PubMed1996

Perturbed interaction between residues 85 and 204 in Tyr-185-->Phe and Asp-85-->Glu bacteriorhodopsins.

According to earlier reports, residue 85 in the bacteriorhodopsin mutants D85E and Y185F deprotonates with two apparent pKa values. Additionally, in Y185F, Asp-85 becomes significantly more protonated during light adaptation. We provide a new explanation for these findings. It is based on the scheme that links the protonation state of residue 85 to the protonation state of residue 204 (S.P. Balashov, E.S. Imasheva, R. Govindjee, and T.G. Ebrey. 1996. Biophys. J. 70:473-481; H.T. Richter, L.S. Brown, R. Needleman, and J.K. Lanyi. 1996. Biochemistry. 35:4054-4062) and justified by the observation that the biphasic titration curves of D85E and Y185F are converted to monophasic when the E204Q residue change is introduced as a second mutation. Accordingly, the D85E and Y 185F mutations are not the cause of the biphasic titration, as that is a property of the wild-type protein. By perturbing the extracellular region of the protein, the mutations increase the pKa of residue 85. This increases the amplitude of the second titration component and makes the biphasic character of the curves more obvious. Likewise, a small rise in the pKa of Asp-85 when the retinal isomerizes from 13-cis, 15-syn to all-trans accounts for the changed titration behavior of Y185F after light adaptation. This mechanism simplifies and unites the interpretation of what had appeared to be complex and unrelated phenomena.

Aspartic Acid↗

PubMed1996

Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle.

The effects of excitation light intensity on the kinetics of the bacteriorhodopsin photocycle were investigated. The earlier reported intensity-dependent changes at 410 and 570 nm are explained by parallel increases in two of the rate constants, for proton transfers to D96 from the Schiff base and from the cytoplasmic surface, without changes in the others, as the photoexcited fraction is increased. Thus, it appears that the pKa of D96 is raised by a cooperative effect within the purple membrane. This interpretation of the wild-type kinetics was confirmed by results with several mutant proteins, where the rates are well separated in time and a model-dependent analysis is unnecessary. Based on earlier results that demonstrated a structural change of the protein after deprotonation of the Schiff base that increases the area of the cytoplasmic surface, and the effects of high hydrostatic pressure and lowered water activity on the photocycle steps in question, we suggest that the pKa of D96 is raised by a lateral pressure that develops when other bacteriorhodopsin molecules are photoexcited within the two-dimensional lattice of the purple membrane. Expulsion of no more than a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pKa.

Bacteriorhodopsins↗

PubMed1996

Reversal of the surface charge asymmetry in purple membrane due to single amino acid substitutions.

Twenty-seven mutant bacteriorhodopsin's were screened to determine the PKa for reversal of the permanent electric dipole moment. The photoelectric response of an aqueous purple-membrane suspension was used to determine the direction of the purple-membrane dipole moment as a function of pH. The pK(a) for the dipole reversal of wild-type bacteriorhodopsin is 4.5. Six of the 27 mutant bacteriorhodopsin's were found to have a pK(a) for dipole reversal larger than that of wild-type bacteriorhodopsin. Two of these mutants, L93T and L93W, involve a neutral amino acid substitution in the interior of the protein. The direction of the purple-membrane permanent electric dipole moment is determined by the purple-membrane surface charge asymmetry. We conclude that these two substitutions, which do not involve charge replacement, alter the pK(a) for the reversal of the purple-membrane surface charge asymmetry. We suggest that these changes to the pK(a) are due to altered protein folding at the surface of the purple-membrane induced by single-site substitutions in the protein interior.

Bacteriorhodopsins↗

PubMed1996

Removal of Mig1p binding site converts a MAL63 constitutive mutant derived by interchromosomal gene conversion to glucose insensitivity.

Maltose fermenting strains of Saccharomyces cerevisiae have one or more complex loci called MAL. Each locus comprises at least three genes: MALx1 encodes maltose permease, MALx2 encodes maltase, and MALx3 encodes an activator of MALx1 and MALx2 (x denotes one of five MAL loci, with x = 1, 2, 3, 4, or 6). The MAL43c allele is constitutive and relatively insensitive to glucose repression. To understand better this unique phenotype of MAL43c, we have isolated several MAL63c constitutive mutants from a MAL6 strain. All constitutive mutants remain glucose repressible, and all have multiple amino acid substitutions in the C-terminal region, now making this region of Mal63cp similar to that of Mal43cp. These changes have been generated by gene conversion, which transfers DNA from the telomeres of chromosome II and chromosome III or XVI to chromosome VIII (MAL6). The removal of a Mig1p binding site from the MAL63c promoter leads to a loss of glucose repression, imitating the phenotype of MAL43c. Conversely, addition of a Mig1p binding site to the promoter of MAL43c converts it to glucose sensitivity. Mig1p modulation of Mal63p and Mal43p expression therefore plays a substantial role in glucose repression of the MAL genes.

Alleles↗

PubMed1995

Glutamic acid 204 is the terminal proton release group at the extracellular surface of bacteriorhodopsin.

We have measured proton release into the medium after proton transfer from the retinal Schiff base to Asp85 in the photocycle and the C = O stretch bands of carboxylic acids in wild type bacteriorhodopsin and the E204Q and E204D mutants. In E204Q, but not in E204D, the normal proton release is absent. Consistent with this, a negative band in the Fourier transform infrared difference spectra at 1700 cm-1 in the wild type, which we now attribute to depletion of the protonated E204, is also absent in E204Q. In E204D, this band is shifted to 1714 cm-1, as expected from the higher frequency for a protonated aspartic than for a glutamic acid. Consistent with their origin from protonated carboxyls, the depletion bands in the wild type and E204D shift in D2O to 1690 and 1703 cm-1, respectively. In the protein structure, Glu204 seems to be connected to the Schiff base region by a chain of hydrogen-bonded water. As with other residues closer to the Schiff base, replacement of Glu204 with glutamine changes the O-H stretch frequency of the bound water molecule near Asp85 that undergoes hydrogen-bonding change in the photocycle. The results therefore identify Glu204 as XH, the earlier postulated residue that is the source of the released proton during the transport, and suggest that its deprotonation is triggered by the protonation of Asp85 through a network that contains water dipoles.

Aspartic Acid↗

PubMed1995

Light-driven chloride ion transport by halorhodopsin from Natronobacterium pharaonis. 1. The photochemical cycle.

The photochemical cycle of the light-driven chloride pump, halorhodopsin from N. pharaonis, is described by transient optical multichannel and single-wavelength spectroscopy in the visible, and in the infrared. Titration of a blue-shift of the absorption maximum upon addition of chloride describes a binding site with a KD of 1 mM. The reaction sequence after the all-trans to 13-cis photoisomerization of the retinal in this chloride binding form is itself dependent on chloride. At 2 M chloride it is described by the scheme: HR-->K<==>L<==>N-->HR that relaxes in a few milliseconds, and is very similar to the photocycle of bacteriorhodopsin under conditions where the retinal Schiff base cannot deprotonate. At lower chloride concentrations, e.g., 0.1 M, however, a red-shifted state termed O appears between N and HR, in equilibrium with N. The absorption spectra of K, L, N, and O are very similar to their counterparts in the bacteriorhodopsin photocycle. As in their equivalents in bacteriorhodopsin, in the N state the retinal is still 13-cis, but it is reisomerized in the O state to all-trans.

Archaea↗

PubMed1995

Light-driven chloride ion transport by halorhodopsin from Natronobacterium pharaonis. 2. Chloride release and uptake, protein conformation change, and thermodynamics.

The photocycle of the light-driven chloride pump, N. pharaonis halorhodopsin, is described by the scheme HR-->K--><==>L<==>N<==>O<==>HR'-->HR. From the chloride dependencies of the rate constants in this model we identify the N-->O and O-->HR' reactions as the steps where chloride release and uptake occur, respectively, during the transport. The dependencies of the rate constants on temperature describe a thermodynamic cycle in which enthalpy-entropy conversion occurs in the O-->HR' reaction. The dependencies of the rate constants on hydrostatic pressure indicate that a substantial volume decrease occurs at the L-->N reaction, a result of a large-scale conformational change. This is the opposite of the volume increase in the photocycle of the proton pump, bacteriorhodopsin, that is implicated in the access change of the active site during the transport and the passage of a proton from the cytoplasmic surface to the active site. The results together suggest a chloride transport mechanism, in which the equivalents of all the ion transfer steps in bacteriorhodopsin occur but in the reverse sense, so as to cause the extracellular-to-cytoplasmic translocation of a chloride ion instead of the cytoplasmic-to-extracellular transport of a proton.

Archaea↗

PubMed1995

Conversion of bacteriorhodopsin into a chloride ion pump.

In the light-driven proton pump bacteriorhodopsin, proton transfer from the retinal Schiff base to aspartate-85 is the crucial reaction of the transport cycle. In halorhodopsin, a light-driven chloride ion pump, the equivalent of residue 85 is threonine. When aspartate-85 was replaced with threonine, the mutated bacteriorhodopsin became a chloride ion pump when expressed in Halobacterium salinarium and, like halorhodopsin, actively transported chloride ions in the direction opposite from the proton pump. Chloride was bound to it, as revealed by large shifts of the absorption maximum of the chromophore, and its photointermediates included a red-shifted state in the millisecond time domain, with its amplitude and decay rate dependent on chloride concentration. Bacteriorhodopsin and halorhodopsin thus share a common transport mechanism, and the interaction of residue 85 with the retinal Schiff base determines the ionic specificity.

Aspartic Acid↗

PubMed1995

Water structural changes at the proton uptake site (the Thr46-Asp96 domain) in the L intermediate of bacteriorhodopsin.

Fourier transform infrared spectra of the L intermediate of light-adapted bacteriorhodopsin were examined for recombinant proteins with amino acid substitutions at Thr46 and Asp96. Two O-H stretching vibrational bands of water, at 3607 and 3577 cm-1, change into stronger H-bonding states in L of the wild type. Thr46-->Val substitution abolished these bands in spite of the fact that [3-18O]threonine-labeling did not shift them, indicating that they correspond to coordination of the water with Thr46. The two water bands were restored, although with changed frequencies, by an additional Asp96-->Asn substitution in Thr46-->Val/Asp96-->Asn. A single Asp96-->Asn substitution abolished the 3607 cm-1 band. Thus, Asp96 also takes part in structural changes in water. The perturbations of these water molecules in the L intermediate displayed a weak correlation with the ratio of intensity change in the two vibrational bands of the Schiff base mode at 1312 and 1301 cm-1 and the rate for the deprotonation of the Schiff base at the L-to-M reaction of the photocycle. We find, therefore, that the water molecules in the cytoplasmic Asp96-Thr46 domain, which comprises the site of proton uptake after formation of the M intermediate, undergo structural changes in the L intermediate already. These changes are transmitted to the extracellular domain and affect interaction of the Schiff base with Asp85, that is far removed from this region.

Aspartic Acid↗

PubMed1995

Interaction of tryptophan-182 with the retinal 9-methyl group in the L intermediate of bacteriorhodopsin.

An intense indole N-H stretching vibrational band at 3486 cm-1 in the difference Fourier transform infrared spectrum is one of the characteristic features of the L intermediate of bacteriorhodopsin [Maeda, Sasaki, Ohkita, Simpson, & Herzfeld (1992) Biochemistry 31, 12543]. This band is now assigned to tryptophan-182. The Trp182-->Phe (W182F) protein shows specific features in the difference spectrum in the visible region upon L formation, and exhibits great delay in the L-M conversion. Fourier transform infrared difference spectra further indicate that while the intensity of the C-methyl in-plane bending vibration at 1009 cm-1 is lost in the L intermediate of the wild type, its intensity remains high in the W182F protein. The intensity of the N-H stretching vibration upon L formation is diminished considerably in an artificial bacteriorhodopsin containing 9-desmethylretinal. It also exhibits delayed M formation. These results suggest that Trp182 interacts with the retinal side chain through the 9-methyl group, and thereby affects the L-to-M conversion.

Bacteriorhodopsins↗

PubMed1995

Functional significance of a protein conformation change at the cytoplasmic end of helix F during the bacteriorhodopsin photocycle.

The second half of the photocycle of the light-driven proton pump bacteriorhodopsin includes proton transfers between D96 and the retinal Schiff base (the M to N reaction) and between the cytoplasmic surface and D96 (decay of the N intermediate). The inhibitory effects of decreased water activity and increased hydrostatic pressure have suggested that a conformational change resulting in greater hydration of the cytoplasmic region is required for proton transfer from D96 to the Schiff base, and have raised the possibility that the reversal of this process might be required for the subsequent reprotonation of D96 from the cytoplasmic surface. Tilt of the cytoplasmic end of helix F has been suggested by electron diffraction of the M intermediate. Introduction of bulky groups, such as various maleimide labels, to engineered cysteines at the cytoplasmic ends of helices A, B, C, E, and G produce only minor perturbation of the decays of M and N, but major changes in these reactions when the label is linked to helix F. In these samples the reprotonation of the Schiff base is accelerated and the reprotonation of D96 is strongly retarded. Cross-linking with benzophenone introduced at this location, but not at the others, causes the opposite change: the reprotonation of the Schiff base is greatly slowed while the reprotonation of D96 is accelerated. We conclude that, consistent with the structure from diffraction, the proton transfers in the second half of the photocycle are facilitated by motion of the cytoplasmic end of helix F, first away from the center of the protein and then back.

Bacteriorhodopsins↗

PubMed1995

Relationship of proton release at the extracellular surface to deprotonation of the schiff base in the bacteriorhodopsin photocycle.

The surface potential of purple membranes and the release of protons during the bacteriorhodopsin photocycle have been studied with the covalently linked pH indicator dye, fluorescein. The titration of acidic lipids appears to cause the surface potential to be pH-dependent and causes other deviations from ideal behavior. If these anomalies are neglected, the appearance of protons can be followed by measuring the absorption change of fluorescein bound to various residues at the extracellular surface. Contrary to widely held assumption, the activation enthalpies of kinetic components, deuterium isotope effects in the time constants, and the consequences of the D85E, F208R, and D212N mutations demonstrate a lack of direct correlation between proton transfer from the buried retinal Schiff base to D85 and proton release at the surface. Depending on conditions and residue replacements, the proton release can occur at any time between the protonation of D85 and the recovery of the initial state. We conclude that once D85 is protonated the proton release at the extracellular protein surface is essentially independent of the chromophore reactions that follow. This finding is consistent with the recently suggested version of the alternating access mechanism of bacteriorhodopsin, in which the change of the accessibility of the Schiff base is to and away from D85 rather than to and away from the extracellular membrane surface.

Bacteriorhodopsins↗