Molecular dynamics of trans-cis isomerization in bathorhodopsin

We report on time-resolved absorption studies of the bovine visual pigment rhodopsin with subpicosecond resolution at room temperature. Our data show that bathorhodopsin, rhodopsin's early photoproduct, is photochemically formed in 3.0 ± 0.7 ps. The data suggest that bathorhodopsin formation is kinetically preceded by two species along the rhodopsin-to-bathorhodopsin reaction coordinate. Several years ago, a number of experiments were attempted to determine the kinetic steps during the rhodopsin to bathorhodopsin transition (2-8). Rhodopsin (Am.. = 498 nm for bovine rhodopsin) and bathorhodopsin (Am.s = 543 nm) differ substantially in their absorption maxima and are, therefore, easily studied by transient absorption spectroscopy. The fastest of these studies, using instrumental time resolution of -6 ps, was not able to resolve the rhodopsin to bathorhodopsin photoconversion at room temperature, although an approximate time of -3 ps was estimated (4, 5). An intriguing result measured at low temperatures (<20 K), where the kinetics are slowed sufficiently to permit measurements with 6-ps resolution on rhodopsin suspended in a water/ethylene glycol glass, showed that bathorhodopsin formation is substantially retarded in rhodopsin where the exchangeable protons are deuterated. Moreover, the kinetics did not follow Arrhenius behavior (3, 9). The large isotope effect and non-Arrhenius behavior were taken as evidence for quantum mechanical tunneling of the proton along the protonated Schiff base hydrogen bond during the photoexcitation process leading to bathorhodopsin.Recent improvements in ultrafast optical pulse generation have made sensitive measurements on subpicosecond time scales possible. Here, we undertake similar studies of the early photochemistry of rhodopsin in both protonated and deuterated aqueous environments at 295 K using subpicosecond pump and probe pulses. Thus, the resolution used here is more than a factor of 10 better than previous measurements. We observe that bathorhodopsin forms in 3.0 ± 0.7 ps in both H20 and 2H20 solvents. Moreover, the results suggest that at least two transient species precede bathorhodopsin formation.MATERIALS AND METHODS Sample Preparation. Bovine rod outer segments were isolated from frozen retinas by the sucrose step gradient as described (5). Samples were washed with 10 mM Hepes (in either H20 or 2H20) and then solubilized in 20 mM n-dodecyl maltoside/0

supporting

confidence: 88%

Ultrafast spectroscopy of the visual pigment rhodopsin.

Yan

Manor

Weng

et al. 1991

“…The effect of protonation on the relative level ordering in the Schiff base compounds is pronounced. The origin of the level ordering reversal was described in the previous section and is correctly predicted by intermediate neglect of differential overlap/partial single and double configuration interaction (INDO-PSDCI) molecular orbital theory (19,20). Comparison of the model chromophore level orderings with that observed in locked-li-cis-rhodopsin (Fig.…”

Section: Resultssupporting

confidence: 52%

“…Thus, the majority of experimental studies have utilized electronic absorption (6)(7)(8)(9)(10), resonance Raman (11,12), nuclear magnetic resonance (13), Fourier-transform IR (14)(15)(16), or picosecond spectroscopy (17,18). The above-cited experimental, as well as theoretical (19)(20)(21)(22)(23)(24), investigations are not in general agreement concerning the state of protonation of the chromophore (see, for example, refs. 2, 3, 5-8, 11-16, and 18-24) or the number and location of the counterions inside the binding site (2, 6-8, 18-21, 23).…”

mentioning

confidence: 99%

Two-photon spectroscopy of locked-11-cis-rhodopsin: evidence for a protonated Schiff base in a neutral protein binding site.

Birge¹,

Murray²,

Pierce³

et al. 1985

133

102

We studied the nature of the protein binding site of rhodopsin, using two-photon spectroscopy to assign the location of the low-lying "covalent" A *.-like 1rir* state in a model rhodopsin containing a locked-li-cis chromophore. The two-photon thermal lens maximum is observed at 22,800 cm-, "2000 cmt above the one-photon absorption maximum, is also proposed. The latter model is interesting because it also accommodates the observed deuterium isotope effect in the form of a proton translocation between the two residues. The translocation is assumed to be a ground state process, initiated subsequent to the photoisomerization of the chromophore and energetically driven via destabilization of the counterion environment as a result of isomerization-induced charge separation.The nature of the protein binding site of rhodopsin is a subject of intense study (1-18) and continued debate (for recent reviews see refs. [1][2][3][4][5]. Despite the efforts of many researchers, well-ordered three-dimensional crystals of rhodopsin have not been prepared, precluding structural analysis using high resolution x-ray diffraction. Thus, the majority of experimental studies have utilized electronic absorption (6-10), resonance Raman (11,12), nuclear magnetic resonance (13), Fourier-transform IR (14-16), or picosecond spectroscopy (17, 18). The above-cited experimental, as well as theoretical (19-24), investigations are not in general agreement concerning the state of protonation of the chromophore (see, for example, refs. 2, 3, 5-8, 11-16, and 18-24) or the number and location of the counterions inside the binding site (2, 6-8, 18-21, 23). With the goal of resolving some of these issues, we report here the two-photon spectrum of locked-ilcis-rhodopsin.The unique diagnostic capabilities of two-photon spectroscopy for studying the binding site of rhodopsin derive in part from the unusual electronic properties of polyenes. [The polyene chromophore of rhodopsin is 11-cis-retinal bound to the opsin protein via a covalent linkage with the E-amino nitrogen of lysine (1)(2)(3)(4)(5) (27). The latter method, which is described in this paper, proved to be more reliable and is based on the previous synthetic work of Akita et al. (8). The incorporation of the locked-11-cis chromophore (shown in Fig. 1) into opsin produces a nonbleachable rhodopsin analog that can withstand the high light fluxes used in two-photon spectroscopy (9, 27). There is compelling evidence to suggest that the "locked" chromophore occupies the same binding site as the native 11-cis chromophore, and the spectroscopic (one-photon absorption and CD) similarities suggest that the counterion environment is unperturbed (8).The following sections describe the generation of the twophoton thermal lens spectrum and the theoretical analysis of the spectroscopic data. Our principal goal is to assign the state of protonation of the chromophore and determine the nature of the local counterion environment. 4117The publication costs of this article were defrayed in part by page c...

“…This was first demonstrated by Warshel (21) who reported a classical trajectory simulation using his "bicycle pedal" model for visual pigments. More recently Birge and co-workers (22,23) have carried out extensive simulation studies on the isomerization dynamics of rhodopsin. Our own analysis, while far less sophisticated, has the advantage of both simplicity and the absence of any detailed assumption as to the exact nature of the motion along the excited-state surface.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence quantum yield of visual pigments: evidence for subpicosecond isomerization rates.

Doukas¹,

Junnarkar²,

Alfano³

et al. 1984

The fluorescence quantum yields (of) for bovine and squid rhodopsins are determined. Both pigments yield similar results, with an average value for o of 1.2 (±0.5)x 10-5. Since the estimated radiative lifetime of rhodopsin is 5 nsec, the rate constant of the process that competes with fluorescence must be on the order of 0.1 psec. Given the large quantum yield for isomerization of rhodopsin's retinal chromophore, this process is likely to correspond to the motion along retinal's C11-C12 torsional coordinate that leads to cistrans isomerization. An empirical excited-state potential energy curve along this coordinate is derived. It is shown that subpicosecond torsional motion to highly twisted nonfluorescing regions of the potential is possible and, in fact, likely. Our results require the existence of a barrier-less excited-state potential energy curve and suggest that cis-trans isomerization occurs in <1 psec.Visual pigments consist of a single chromophore, 11-cis retinal, covalently bound in the form of a protonated Schiff base to the F-amino group of lysine-321 in the apoprotein opsin (for a recent review, see ref. 1). The visual process is initiated by a photochemical event in which rhodopsin is transformed into its primary photoproduct, bathorhodopsin. It has been well-established that the rhodopsin to bathorhodopsin transition is driven by an 11-cis to all-trans photoisomerization of the in situ retinal chromophore. Given the central importance of this process, great efforts have been invested in its detailed characterization.The potential energy curve along the C11-C12 torsional coordinate that connects the rhodopsin and bathorhodopsin ground states can be described with considerable accuracy (2, 3). There is an unusually large enthalpy increase, the value of which has been determined by Cooper to be 35 kcal/ mol (1 cal = 4.184 J) (3). Based on this value and the absence of a thermal back-reaction at 77 K, a lower limit for the activation energy of 42 kcal/mol can be established (see below). As we discuss below, the upper limit is at most a few kcal/ mol higher than this value. The unusual shape of the groundstate potential is due to specific protein-chromophore interactions designed to enhance the stability of rhodopsin to thermal isomerization and to produce the high energy photoproduct bathorhodopsin (2).The excited-state potential energy surface is clearly designed to facilitate an efficient 11-cis to all-trans photoisomerization (2). The quantum yield for the process is, indeed, unusually high (0.67), and this value remains constant at temperatures as low as 77 K (4). Moreover, the primary event is extremely fast, occurring on picosecond, or perhaps subpicosecond, time scales (5-8). In light of the fact that protonated Schiff bases in solution do not exhibit such unusual photolability (9), it is of interest to determine how the properties of the chromophore are so drastically altered on binding to the protein. Based on an analysis of the photochemistry, it was proposed that rhodopsin and batho...