Conformational change of the E-F interhelical loop in the M photointermediate of bacteriorhodopsin

The conformation of the structured EF interhelical loop of bacteriorhodopsin and its change in the M photointermediate were assessed by measuring the rate of reaction of 16 single engineered cysteine residues along the loop with water-soluble sulfhydryl reagents. The exposure to the bulk in the unilluminated state determined with the cysteine reaction correlated well with the degree of access to water calculated from the crystallographic structure of the loop. The EF-loop should be affected by the well-known outward tilt of helix F in the M and N intermediates of the photocycle. A second mutation in each cysteine mutant, the D96N residue replacement, allowed full conversion to the M state by illumination. The reaction rates measured under these conditions indicated that buried residues tend to become more exposed, and exposed residues become more buried in M. This is to be expected from tilt of helix F. However, the observation of increased exposure of four residues near the middle of the loop, where steric effects are only from other loop residues, indicate that the conformation of the EF-loop itself is changed. Thus, the motion of the loop in M is more complex than expected from simple tilt of helix F, and may include rotation that unwinds its twist.     

Structural changes in the L photointermediate of bacteriorhodopsin

The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53-1.73 A resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C(13)=C(14) and C(15)=NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L-->M reaction is likely to occur, therefore, via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.     

Electron paramagnetic resonance study of structural changes in the O photointermediate of bacteriorhodopsin

The structural changes of bacteriorhodopsin during its photochemical cycle, as revealed by crystal structures of trapped intermediates, have provided insights to the proton translocation mechanism. Because accumulation of the last photointermediate, O, appears to be hindered by lattice forces in the crystals, the only information about the structure of this state is from suggested analogies with the determined structures of the non-illuminated D85S mutant and wild-type bacteriorhodopsin at low pH. We used electron paramagnetic resonance spectroscopy of site-directed spin labels at the extracellular protein surface in membranes to test these models. Spin-spin dipolar interactions in the authentic O state compared to the non-illuminated state revealed that the distance between helices C and F increases by ca 4 Angstroms, there is no distance change between helices D and F, and the distance between helix D and helix B of the adjacent monomer increases. Further, the mobility changes of single labels indicate that helices E and F move outward from the proton channel at the center of the protein, and helix D tilts inward. The overall pattern of movements suggests that the model at acid pH is a better representation of the O state than D85S. However, the mobility analysis of spin-labels on the B-C interhelical loop indicates that the antiparallel beta-sheet maintains its ordered secondary structure in O, instead of the predicted disorder in the two structural models. During decay of the O state, the last step of the photocycle, a proton is transferred from Asp85 to proton release complex in the extracellular proton channel. The structural changes in O suggest the need of large conformational changes to drive the Arg82 side-chain back to its initial orientation towards Asp85, and to rearrange the numerous water molecules in this region in order to conduct the proton away from Asp85.     

Water-mediated hydrogen-bonded network on the cytoplasmic side of the Schiff base of the L photointermediate of bacteriorhodopsin

The L intermediate in the proton-motive photocycle of bacteriorhodopsin is the starting state for the first proton transfer, from the Schiff base to Asp85, in the formation of the M intermediate. Previous FTIR studies of L have identified unique vibration bands caused by the perturbation of several polar amino acid side chains and several internal water molecules located on the cytoplasmic side of the retinylidene chromophore. In the present FTIR study we describe spectral features of the L intermediate in D(2)O in the frequency region which includes the N-D stretching vibrations of the backbone amides. We show that a broad band in the 2220-2080 cm(-1) region appears in L. By use of appropriate (15)N labeling and mutants, the lower frequency side of this band in L is assigned to the amides of Lys216 and Gly220. These amides are coupled to each other, and interact with Thr46 and Val49 in helix B and Asp96 in helix C via weakly H-bonding water molecules that exhibit O-D stretching vibrations at 2621 and 2605 cm(-1). These water molecules are part of a hydrogen-bonded network characteristic of L which includes other water molecules located closer to the chromophore that exhibit an O-D stretching vibration at 2589 cm(-1). This structure, extending from the Schiff base to the internal proton donor Asp96, stabilizes L and affects the L-to-M transition.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.     

Photoreaction of bacteriorhodopsin at high pH: origins of the slow decay component of M.

The absorption spectrum of light-adapted purple membrane in 3 M KCl is dependent on temperature even in the room temperature region. Temperature-induced difference spectra at various pH values suggested that the trans isomer of bacteriorhodopsin, bR570, is in thermal and/or photodynamic equilibrium with several different conformers. The major second conformer occurring at neutral pH had the same spectroscopic properties as the 13-cis isomer, and its content at 35 degrees C was estimated to be more than 20%. Heterogeneity in the protein conformation became more significant above pH8, where temperature-induced difference spectra exhibited a negative peak at 580 nm and a positive peak at 296 nm. This absorption change is very similar to that observed upon the formation of the N intermediate, suggesting that an N-like conformer occurs at high pH and temperature. A significant temperature dependence was also seen in the M decay kinetics at high pH, which were described by two decay components; i.e., the fast decaying M (Mf) was predominant at low temperature, but the amplitude of the slow component (M(s)) increased with increasing temperature. It is suggested that M(s) is generated upon excitation of the N-like conformer, in which the residue (Asp-96) usually acting as a proton donor to the Schiff base is deprotonated. The N-like conformer could be N itself, because M(s) was enhanced when N was accumulated by background light. A strong correlation between the amplitude of M(s) and the concentration of N was also revealed by the accumulation kinetics of Mf, M(s), and N after the onset of continuous actinic light.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathways of proton release in the bacteriorhodopsin photocycle.

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.     

Generation of the O630 photointermediate of bacteriorhodopsin is controlled by the state of protonation of several protein residues

The last stages of the photocycle of the photosynthetic pigment all-trans bacteriorhodopsin (bR570), as well as its proton pump mechanism, are markedly pH dependent. We have measured the relative amount of the accumulated O630 intermediate (Phir), as well as its rise and decay rate constants (kr and kd, respectively), over a wide pH range. The experiments were carried out in deionized membrane suspensions to which varying concentrations of metal cations and of large organic cations were added. The observed pH dependencies, s-shaped curves in the case of Phir and bell-shaped curves for kr and kd, are interpreted in terms of the titration of three protein residues denoted as R1, R2, and R3. The R1 titration is responsible for the increase in Phir, kr, and kd upon lowering the pH from pH approximately 9.5 to 7. At low pH Phir exhibits a secondary rise which is attributed to the titration of a low pKa group, R2. After reaching a maximum at pH approximately 7, kr and kd undergo a decrease upon decreasing the pH, which is attributed to the titration of R3. All three titrations exhibit pKa values which decrease upon increasing the salt concentration. As in the case of the Purple (bR570) if Blue (bR605) equilibrium, divalent cations are substantially more effective than monovalent cations in shifting the pKa values. Moreover, bulky organic cations are as effective as small metal cations. It is concluded that analogously to the Purple if Blue equilibrium, the salt binding sites which control the pKa values of R1, R2, and R3 are located on, or close to, the membrane surface. Possible identifications of the three protein residues are considered. Experiments with the E204Q mutant show that the mutation has markedly affected the R2 (Phir) titration, suggesting that R2 should be identified with Glu-204 or with a group whose pKa is affected by Glu-204. The relation between the R1, R2 and R3 titrations and the proton pump mechanism is discussed. It is evident that the pH dependence of Phir is unrelated to the measured pKa of the group (XH) which releases the proton to the extracellular medium during the photocycle. However, since the same residue may exhibit different pKa values at different stages of the photocycle, it cannot be excluded that R2 or R3 may be identified with XH.     

Coupling of the reisomerization of the retinal, proton uptake, and reprotonation of Asp-96 in the N photointermediate of bacteriorhodopsin

In the N to O reaction of the bacteriorhodopsin photocycle, Asp-96 is protonated from the cytoplasmic surface, and coupled to this, the retinal isomerizes from 13-cis,15-anti back to the initial all-trans configuration. To dissect the two steps, and to better understand how and why they occur, we describe the properties of two groups of site-specific mutants in which the N intermediate has greatly increased lifetime. In the first group, with the mutations near the retinal, an unusual N state is produced in which the retinal is 13-cis,15-anti but Asp-96 has a protonated carboxyl group. The apparent pK(a) for the protonation is 7.5, as in the wild-type. It is likely that here the interference with N decay is the result of steric conflict of side-chains with the retinal or with the side-chain of Lys-216 connected to the retinal, which delays the reisomerization after protonation of Asp-96. In the second group, with the mutations located near Asp-96 or between Asp-96 and the cytoplasmic surface, reprotonation of Asp-96 is strongly perturbed. The reisomerization of the retinal occurs only after recovery from a long-living protein conformation in which reprotonation of Asp-96 is either entirely blocked or blocked at low pH.     

Polarized Fourier transform infrared (FTIR) difference spectroscopy of the M412 intermediate in the bacteriorhodopsin photocycle

The possibility that light-induced protein conformational changes accompany the formation of the M₄₁₂ species in the bacteriorhodopsin photocycle is investigated by polarized Fourier transform infrared (FTIR) spectroscopy on oriented films of purple membrane. From the light-induced FTIR dichroism changes, it is estimated that: (i) the CO stretching vibration at 1762 cm⁻¹, which has been assigned to a protonated Asp carboxyl group in M₄₁₂ [(1985) Biochemistry 24, 400-407], is oriented at (θ = 35 ± 5° from the normal to the membrane plane; (ii) the limit for the change in the average tilt angle of the α-helices after photoconversion is less than 2°. The latter observation excludes the large variations in the protein conformation during the M⁴¹² formation proposed by Draheim and Cassim [(1985) Biophys. J. 47, 497-507].     

Pathways modulating neural KCNQ/M (Kv7) potassium channels

K(+) channels play a crucial role in regulating the excitability of neurons. Many K(+) channels are, in turn, regulated by neurotransmitters. One of the first neurotransmitter-regulated channels to be identified, some 25 years ago, was the M channel. This was categorized as such because its activity was inhibited through stimulation of muscarinic acetylcholine receptors. M channels are now known to be composed of subunits of the Kv7 (KCNQ) K(+) channel family. However, until recently, the link between the receptors and the channels has remained elusive. Here, we summarize recent developments that have begun to clarify this link and discuss their implications for physiology and medicine.     

Formation of the M(N) (M(open)) intermediate in the wild-type bacteriorhodopsin photocycle is accompanied by an absorption spectrum shift to shorter wavelength, like that in the mutant D96N bacteriorhodopsin photocycle

Maximum of the M intermediate difference spectrum in the wild-type Halobacterium salinarium purple membrane is localized at 405-406 nm under conditions favoring accumulation of the M(N) intermediate (6 M guanidine chloride, pH 9.6), whereas immediately after laser flash the maximum is localized at 412 nm. The maximum is also localized at 412 nm 0.1 msec after the flash in the absence of guanidine chloride at pH 11.3. Within several milliseconds the maximum is shifted to short-wavelength region by 5-6 nm. This shift is similar to that in the D96N mutant which accompanies the M(N) (M(open)) intermediate formation. The main two differences are: 1) the rate of the shift is slower in the wild-type bacteriorhodopsin, and is similar to the rate of the M to N intermediate transition (t1/2 approximately 2 msec); 2) the shift in the wild-type bacteriorhodopsin is observed at alkaline pH values which are higher than pK of the Schiff base (approximately 10.8 at 1 M NaCl) in the N intermediate with the deprotonated Asp-96. Thus, the M(N) (M(open)) intermediate with open water-permeable inward proton channel is observed only at high pH, when the Schiff base and Asp-96 are deprotonated. The data confirmed our earlier conclusion that the M intermediate observed at lower pH has the closed inward proton channel.     

Structural changes of the complex between pharaonis phoborhodopsin and its cognate transducer upon formation of the M photointermediate

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes, and the association is weakened by 2 orders of magnitude in the M intermediate. Such change is believed to correspond to the transfer of the light signal to pHtrII. In this paper, we applied Fourier transform infrared (FTIR) spectroscopy to the active M intermediate in the absence and presence of pHtrII. The obtained difference FTIR spectra were surprisingly similar, notwithstanding the presence of pHtrII. This result strongly suggests that the transducer activation in the ppR-pHtrII system does not induce secondary structure alterations of the pHtrII itself. On the other hand, we found that the hydrogen bond of the OH group of Thr204 is altered in the primary K intermediate, but restored in the M intermediate. The hydrogen bond of Asn74 in pHtrII is strengthened in M, presumably because of the change in interaction with Tyr199 of ppR. These facts provided a light signaling pathway from Lys205 (retinal) of the receptor to Asn74 of the transducer through Thr204 and Tyr199. Transducer activation is likely to involve a relaxation of Thr204 in the receptor and hydrogen bonding alteration of Asn74 in the transducer, during which the helices of the transducer perform rigid-body motion without changing their secondary structures.     

C(13)-substituted bacteriorhodopsin analogs

13-Ethyl-, 13-isopropyl-, 13-tert-butyl-, 13-phenyl-, 13-alpha-naphthyl-, and 13-demethyl-retinals were synthesized and incubated with bacterioopsin (bO) to give the corresponding bacteriorhodopsin (bR) analogs. The capability of the 13-tert-butyl- and 13-alpha-naphthyl-bRs to exist and to photocycle shows that apparently around C(13) of the chromophore there lies a large enough cavity. A study of the light-induced conversions of the artificial pigments prepared has shown that the introduction at position 13 of the chromophore of the hydrocarbon substituents bulkier than that of the natural bR diminished the amplitudes of the electric photoresponses. Bulky C(13)-substituents or absence of substitution at that position decelerated the relaxation of the M-intermediates and disturbed the 13-cis-in equilibrium all-trans-isomerization.     

Structure of rhodopsin and the metarhodopsin I photointermediate

The structure of the visual pigment rhodopsin in the dark state was first investigated by electron microscopy (EM). More recently, rhodopsin has been crystallised in two different space groups--a tetragonal P4(1) crystal form and a trigonal P3(1) packing arrangement. The structures of the pigment, determined by X-ray crystallography from these two crystal forms, show many similarities, but also significant differences. These differences are most extensive in the G-protein-binding region of the cytoplasmic surface, where the location of the loop between helices 5 and 6 is highly variable. A combination of EM and spin labelling suggests that this loop adopts the native conformation in the P3(1) crystal form. The X-ray structures also show the location of structural water molecules that are important for colour tuning, stabilisation of the ground state and receptor activation, and act as a template for modelling other G-protein-coupled receptors. A major current focus of structural work on rhodopsin is investigation of the activated state of the receptor. After careful spectroscopic characterisation of light activation in two-dimensional crystals, a map of the metarhodopsin I intermediate was obtained by EM from two-dimensional crystals. In addition, NMR studies are providing information about the structure of activated states of rhodopsin. In the future, structural information will show how rhodopsin becomes activated and how it couples to downstream signalling pathways.     

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.     

Electron diffraction analysis of the M412 intermediate of bacteriorhodopsin.

High resolution electron diffraction data have been recorded for glucose-embedded purple membrane specimens in which bacteriorhodopsin (bR) has been trapped by cooling slowly to below--100 degrees C under continuous illumination. Thin films (OD approximately 0.7) of glucose-embedded membranes, prepared as a control, showed virtually 100% conversion to the M state, and stacks of such thin film specimens gave very similar x-ray diffraction patterns in the bR568 and the M412 state in most experiments. To be certain that any measured differences in diffraction intensity would be real, two independent sets of electron diffraction intensities were recorded for near-equatorial, i.e. (hkO), reflections. Little correlation was indeed observed between these two sets for delta F values at low resolution (15-5.0 A, 49 reflections), but the correlation coefficient is approximately 0.3 at high resolution (5.0-3.3 A, 218 reflections). Thus, while most of the measured difference is error, the mean delta F and the correlation coefficient can be used to estimate the smaller, true delta F due to structural changes occurring in the M state. The magnitude of this estimated true mean delta F is equal to what would be produced if approximately five to seven nonhydrogen atoms were moved to structurally uncorrelated (i.e., new) positions in the M state. Movements of a few amino acid side chains, and repositioning of atoms of the retinal group and the associated lysine side chain after trans-cis isomerization, are the most probable causes of the observed intensity changes in the M state.(ABSTRACT TRUNCATED AT 250 WORDS)     

(M,R)-systems, (P,M,C)-nets, hierarchical decay, and biological aging: reminiscences of Robert Rosen

I have the pleasure to present a number of personal experiences that I had with Robert Rosen, both as his student and as a research colleague, and I will describe how this affected my academic career over the past decades. As a matter of fact, Rosen's work with (M,R)-systems as well as his continuing mentorship guided me into my own research in gerontology and geriatrics. Amazingly, this still continues to affect my work in complexity theory after 30 years.     

Nature of the principal photointermediate of halorhodopsin

Two alternative hypotheses have been presented as to the nature of the principal halorhodopsin photointermediate: a) it is a form whose its absorption band is shifted from the 575 nm position to 500 or 520 nm, and b) it is a form whose absorption band is shifted to only about 565 nm, but with an altered band shape so it exhibits a fortuitous difference peak near 500 nm. Such a shift with a maximum near 500 nm is also obtained in the dark when chloride is removed from the sample, suggesting the hypothesis that the spectral changes reflect the transient detachment of chloride from a binding site (Ogurusu et al, J. Biochem. Tokyo 95, 1073-1082, 1984). Comparison of the quantum yields of flash-induced absorption changes in halorhodopsin and bacteriorhodopsin strongly suggests, however, that hypothesis b) is untenable.