Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids

The effect of lipid ordering on the kinetics and extent of metarhodopsin II (meta II) formation was evaluated in bovine rhodopsin which had been reconstituted into phosphatidylcholine vesicles containing 0, 15, and 30 mol% cholesterol. The rate of establishment of the dynamic equilibrium between metarhodopsin I (meta I) and the two kinetically distinguished forms of meta II in the branched meta II model [meta IIfast and meta IIslow; Straume, M., Mitchell, D. C., Miller, J. L., & Litman, B. J. (1990) Biochemistry (preceding paper in this issue)] is derived from kinetic measurements of rhodopsin photolysis in these vesicle systems at several temperatures. Values of the meta I in equilibrium with meta IItotal equilibrium constant, Keq, are calculated from the derived model-dependent rate constants, and are shown to be equivalent to those derived from rapidly acquired absorbance spectra. The presence of 30 mol% cholesterol reduces Keq by approximately 50% between 10 and 37 degrees C. Analysis of the model-dependent parameters in terms of delta H and delta S reveals that cholesterol raises the free energy of meta IIslow, relative to meta I, by increasing delta H whereas it raises the relative free energy of meta IIfast by making delta S meta IIfast relative to meta I less positive. The reduction in Keq by both temperature and cholesterol is found to be directly correlated with a parameter that reflects the free volume available for molecular motion in the hydrophobic core of the bilayer [Straume, M., & Litman, B. J. (1988) Biochemistry 27, 7723-7733].(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhodopsin in dimyristoylphosphatidylcholine-reconstituted bilayers forms metarhodopsin II and activates Gt

The photochemical intermediate metarhodopsin II (meta II; lambda max = 380 nm) is generally identified with rho*, the conformation of photolyzed rhodopsin which binds and activates the visual G-protein, Gt [Emeis, D., & Hoffman, K.P. (1981) FEBS Lett. 136, 201-207]. Purified bovine rhodopsin was incorporated into vesicles consisting of dimyristoylphosphatidylcholine (DMPC), and the rapid formation of a photochemical intermediate absorbing maximally at 380 nm was quantified via both flash photolysis and equilibrium spectral measurements. Kinetic and equilibrium spectral measurements performed above the Tm of DMPC showed that Gt, in the absence of GTP, enhances the production of the 380-nm-absorbing species while reducing the concentration of the 478-nm-absorbing species, metarhodopsin I (meta I), in a manner similar to that observed in the native rod outer segment disk membrane. This Gt-induced shift in the equilibrium concentration of photointermediates indicated that the species with an absorbance maximum at 380 nm was meta II. The presence of rho* in the DMPC bilayer was established via measurements of photolysis-induced exchange of tritiated GMPPNP, a nonhydrolyzable analogue of GTP, on Gt. Above Tm, the metarhodopsin equilibrium is strongly shifted toward meta I relative to the native rod outer segment disk membrane; however, at 37 degrees C, 40% of the photointermediates are in the form of meta II. The formation of meta II above Tm is slowed by a factor of ca. 2 relative to the disk membrane. Below Tm, the equilibrium is shifted still further toward meta I, and meta II forms ca. 7 times slower than in the disk membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature and pH dependence of the metarhodopsin I-metarhodopsin II kinetics and equilibria in bovine rod disk membrane suspensions

The kinetics of the relaxation of bleached bovine rod disk membrane suspensions from metarhodopsin I into the equilibrium between metarhodopsins I and II were determined at pHs between 5.9 and 8.1 and at temperatures between -1 and 15 degrees C. From these data, thermodynamic equations were generated by two-way linear regression that simultaneously describe the functional dependence on pH and temperature of the pseudo-first-order and true forward rate constants, the reverse and observed rate constants, and the equilibrium constant. Using these equations, we obtained the thermodynamic parameters and the apparent net proton uptake for the transitions from metarhodopsin I to metarhodopsin II and from metarhodopsin I to the activated intermediate. The reversibility of this equilibrium and the effect of aging of the preparation on the measured rate constants were investigated.     

Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium.

The effect of phospholipid bilayer acyl chain packing free volume on the equilibrium concentration of the form of photolyzed rhodopsin which initiates visual signal transduction, metarhodopsin II (meta II), is examined in reconstituted systems formed from the saturated phospholipid dimyristoylphosphatidylcholine (DMPC) and in the polyunsaturated phospholipid sn-1-palmitoyl-sn-2-arachidonoylphosphatidylcholine (PAPC) with and without 30 mol% cholesterol. The extent of meta II formation is determined from both flash photolysis measurements and rapidly acquired absorbance spectra. Equilibrium and dynamic properties of the lipid bilayer are characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH orientational properties are characterized by fv, a parameter which reflects the volume available for probe reorientation in the bilayer, relative to that available in an unhindered, isotropic environment [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5121-5126]. The metarhodopsin I in equilibrium with meta II equilibrium constant, Keq has a linear relationship with fv for rhodopsin in PAPC vesicles with and without cholesterol as well as for rhodopsin in DMPC vesicles, and these two correlation lines have different slopes. The correlations between Keq and fv in PAPC and DMPC systems are compared with a similar correlation in the native rod outer segment disk membrane and one reported previously in an egg phosphatidylcholine (egg PC) system [Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990) Biochemistry 29, 9143-9149].(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-induced interaction between rhodopsin and the GTP-binding protein. Metarhodopsin II is the major photoproduct involved

We have previously described [H, Kühn et al. (1981) Proc. Natl Acad. Sci. USA, 78, 6873-6877] a light-induced scattering change ('binding signal') associated with a stoichiometric binding between photoexcited rhodopsin and a peripheral membrane protein, the GTP-binding protein, in bovine rod outer segment suspensions. We have attempted here to identify the rhodopsin intermediate R* which is responsible for this interaction, by studying its dependence on pH, temperature and ionic strength. The results strongly suggest that the active state is metarhodopsin II (M II). 1. The initial phase of the binding signal is slightly slower than the formation of metarhodopsin II (2-37 degrees C, pH 5.5-9). 2. The kinetics of the decay of the active rhodopsin state are similar to those of the metarhodopsin II leads to metarhodopsin III transition (37 degrees C, pH 7.3). 3. All conditions which lead to light-induced binding of the GTP-binding protein to R* also lead to the formation of M II. At 2 degrees C, pH 8.3, in particular where no M II is formed in the absence of GTP-binding protein, binding signals and light-induced attachment of the GTP-binding protein to the membrane are still observed. Consistently, addition of GTP-binding protein to a suspension of extracted membranes bleached at 2 degrees C (pH 8.3) shifts the metarhodopsin I in equilibrium metarhodopsin II equilibrium towards metarhodopsin II. The shift is reversed by GTP, which dissociates the rhodopsin--GTP-binding protein complex. 4. At low ionic strength, where the GTP-binding protein is soluble in the dark (instead of being associated to the membrane as in the above experiments) M II still induces the binding whereas M I does not, indicating a much lower affinity of the GTP-binding protein for MI.     

Photolysis intermediates of human rhodopsin.

Photochemical studies were conducted on human rhodopsin at 20 degrees C to characterize the intermediates which precede the formation of metarhodopsin II, the trigger for the enzyme cascade mechanism of visual transduction. Human rhodopsin was prepared from eyes which had previously been used for corneal donations. Time resolved absorption spectra collected from 10(-8) to 10(-6) s after photolysis of human rhodopsin in detergent suspensions displayed biexponential decay kinetics. The apparent lifetimes obtained from the data are 65 +/- 20 and 292 +/- 25 ns, almost a factor of 2 slower than the corresponding rates in bovine rhodopsin. The spectra can be fit well using a model in which human bathorhodopsin decays toward equilibrium with a blue-shifted intermediate (BSI) which then decays to lumirhodopsin. Spectra and kinetic rate constants were determined for all these intermediates using a global analysis which showed that the spectra of the human intermediates are remarkably similar to bovine intermediates. Microscopic rate constants derived from this model are 7.4 x 10(6) s-1 for bathorhodopsin decay and 7.5 x 10(6) s-1 and 4.6 x 10(6) s-1 for the forward and reverse reactions of BSI, respectively. Decay of lumirhodopsin to later intermediates was studied from 10(-6) to 10(-1) s after photolysis of rhodopsin in human disk membrane suspensions. The human metarhodopsin I in equilibrium metarhodopsin II equilibrium appears to be more forward shifted than in comparable bovine studies.     

Euphausiid visual pigments. The rhodopsins of Euphausia superba and Meganyctiphanes norvegica (Crustacea, Euphausiacea)

The rhabdoms of Euphausia superba contain one digitonin-extractable rhodopsin, lambda max 485 nm. The rhodopsin undergoes unusual pH- dependent spectral changes: above neutrality, the absorbance decreases progressively at 485 nm and rises near 370 nm. This change is reversible and appears to reflect an equilibrium between a protonated and an unprotonated form of the rhodopsin Schiff-base linkage. Near neutral pH and at 10 degrees C, the rhodopsin is partiaLly converted by 420-nm light to a stable 493-nm metarhodopsin. The metarhodopsin is partially photoconverted to rhodopsin by long-wavelength light in the absence of NH2OH; in the presence of NH2OH, it is slowly converted to retinal oxime and opsin. The rhodopsin of Meganyctiphanes norvegica measured in fresh rhabdoms by microspectrophotometry has properties very similar to those of the extracted rhodopsin of E. superba. Its lambda max is 488 nm and it is partially photoconverted by short wavelength irradiation to a stable photoconvertible metarhodopsin similar to that of E. superba. In the presence of light and NH2OH, the M. norvegica metarhodopsin is converted to retinal oxime and opsin. Our results indicate that previous determinations of euphausiid rhodopsin absorbance spectra were incorrect because of accessory pigment contamination.     

Interplay between hydroxylamine,metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes

The decay reactions of metarhodopsin II and the dissociation of the complex between rhodopsin (in the metarhodopsin II state) and the GTP-binding protein (G-protein) (in its inactive, GDP-binding form) have been compared at various concentrations of hydroxylamine. The reactions of the chromophore were measured by absorption changes in the visible range, the complex dissociation by changes in the near-in-frared scattering. An additional monitor of the complex was given by the G-protein-dependent equilibrium between metarhodopsin I and metarhodopsin II. For all measurements, fragments of isolated bovine rod outer segments in suspension were used. In the absence of hydroxylamine, the rhodopsin-G-protein complex dissociated within 20–30 min at room temperature. The presence of hydroxylamine greatly accelerated (e.g., 5-fold at 1 mM NH₂OH) the dissociation. Under all conditions, the free, dissociated G-protein can reassociate to metarhodopsin II produced by subsequent bleaching. Dissociation of the metarhodopsin II-G-protein complex required the decay of photoproducts with a maximal absorbance of 380 nm, but was not affected by the simultaneous presence of metarhodopsin III or metarhodopsin III — like photoproducts with a maximal absorbance between 450 and 470 nm. Despite the acceleration of metarhodopsin II-G-protein dissociation by NH₂OH, metarhodopsin II-G-protein was relatively stabilized as compared to free metarhodopsin II. The ratio of the decay rates of free metarhodopsin II and metarhodopsin III-G-protein was increased as much as 10-fold in the presence of 25 mM NH₂OH. The results indicate a mutual interdependence of retinal, opsin and G-protein.     

Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation.

Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.     

Studies on cephalopod rhodopsin: photoisomerization of the chromophore.

Photoisomerization of the chromophore of squid rhodopsin is dependent upon the irradiation temperature. Above 0 degrees C, only 11-cis in equilibrium all-trans reaction proceeds and the all-trans leads to 9-cis reaction is limited to extremely low efficiency. At liquid nitrogen temperature, 11 cis in equilibrium all-trans in equilibrium 9-cis reaction takes place. At intermediary low temperatures (-80 degrees C to -15 degrees C) another isomer of retinal may be produced by the irradiation, which forms a pigment having an absorbance maximum at 465 nm (P-465). The formation of P-465 decreases remarkably in the narrow temperature range from -30 degrees C to 0 degrees C where mesorhodopsin converts to metarhodopsin. Medsorhodopsin is quite different from metarhodopsin in the photoisomerization of the chromophore because P-465 is produced from the former but not from the latter. No P-465 is produced both at liquid nitrogen temperature and above 0 degrees C. P-465 is more labile than any of the other photoproducts so far known, that is isorhodopsin, alkaline and acid metarhodopsins. P-465 is converted to metarhodopsin by irradiation.     

Bleaching kinetics of artificial visual pigments with modifications near the ring-polyene chain connection

Absorbance difference spectra were recorded at 20 degrees C from 30 ns to milliseconds after photolysis of lauryl maltoside suspensions of artificial visual pigments derived from 9-cis isomers of 5-ethylretinal, 8,16-methanoretinal (a 6-s-trans-bicyclic analogue), or 5-demethyl-8-methylretinal. In all three pigments, the earliest intermediate that was detected had the characteristics of a mixture of bathorhodopsin and a blue-shifted intermediate, BSI, which is the first decay product of bathorhodopsin in bovine rhodopsin. The first decays resolved on the nanosecond time scale were the formation of the lumirhodopsin analogues. Subsequent decays were able to be fit with a mechanistic scheme which has been shown to apply to both membrane and detergent suspensions of rhodopsin. Large increases were seen in the amount of metarhodopsin I which appeared after photolysis of 5-ethylisorhodopsin and the bicyclic isorhodopsin analogue, while 5-demethyl-8-methylisorhodopsin more closely followed native rhodopsin in decaying through meta I380, a 380 nm absorbing precursor to metarhodopsin II. In addition to forming more metarhodopsin I, the bicyclic analogue stabilized the metarhodopsin I-metarhodopsin II equilibrium similarly to what has been previously reported for 9-demethylrhodopsin in detergent, introducing the possibility that the bicyclic analogue could similarly be defective in transducin activation. These observations support the idea that long after initial photolysis, structural details of the retinylidene chromophore continue to play a decisive role in processes leading to the activated form, metarhodopsin II.     

Functional equivalence of metarhodopsin II and the Gt-activating form of photolyzed bovine rhodopsin

Absorption of a photon by the visual pigment rhodopsin leads to the formation of an activated conformational state, denoted rho*, which is capable of activating the visual G-protein, Gt. The bleaching of rhodopsin can be resolved into a series of spectrally distinct photointermediates. Previous studies suggest that the photointermediate metarhodopsin II (meta II, lambda max of 380 nm) corresponds to the physiologically active form rho*. In the studies reported herein, spectral and enzymological data were analyzed and compared so as to evaluate the temporal correspondence between meta II and rho*. This information was obtained by direct observation of the meta II and rho* decay times in parallel experiments utilizing identical preparations of urea-stripped, bovine retinal rod outer segment disk membranes at pH 8.0, 20 degrees C. Postflash spectra were deconvolved to resolve the meta II absorbance at 380 nm, and a decay time for the loss of meta II of 8.2 min (SD = 0.5 min) was obtained from fitting these data to a single-exponential decay process. The diminishing ability of bleached rhodopsin to activate Gt was measured by monitoring the level of catalyzed exchange of Gt-bound GDP for a nonhydrolyzable GTP analogue. Analysis of the decrease in the initial velocity of nucleotide exchange, measured at various postflash incubation times, yielded a rho* decay time of 7.7 min (SD = 0.5 min) when analyzed as a single-exponential process. The similarity of these decay times provides direct evidence that meta II and rho* are present over the same time regime, and further supports the equivalence of these two forms of photoactivated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical aspects of the visual process. XL. Spectral and chemical analysis of metarhodopsin III in photoreceptor membrane suspensions

The late photointermediates of rhodopsin photolysis have been analyzed spectrally and chemically in bovine rod outer segment membrane suspension at 25 degrees C and pH 6.5. The decay of metarhodopsin II follows two spectrally distinct routes, resulting 40 min after illumination in a stable mixture of photo-products with absorbance maxima around 380 and 452 nm, free retinal and metarhodopsin III, respectively. Chemical analysis shows that three different products are involved: free retinal (approx. 34%), protein-bound retinal (approx. 51%) and lipid-bound retinal (approx. 15%). The latter fraction consists of retinylidene-phosphatidylethanolamine exclusively. Photolysis of membranes reconstituted with various phospholipids gives a qualitatively normal spectral picture, but the production of metarhodopsin III may vary with the phospholipid composition, i.e. with the percent of phosphatidylethanolamine present. Chemical analysis shows that with increasing phosphaatidylethanolamine content of the membrane, the retinylidene phosphatidylethanolamine fraction increases proportionally at the expense of free retinal, while the fraction of protein-bound retinal remains unaffected. The results indicate that under these conditions metarhodopsin III (defined as a long wavelength product of metarhodopsin II decay) is composed of two chemically distinct components: opsin-bound retinal and retinylidene phosphatidylethanolamine.     

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (λ > 520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.     

Effects of lipid environment on the light-induced conformational changes of rhodopsin. 1. Absence of metarhodopsin II production in dimyristoylphosphatidylcholine recombinant membranes

Photolysis of bovine rhodopsin in dimyristoylphosphatidylcholine recombinant membranes results in the production of a relatively stable metarhodopsin I like photointermediate that decays slowly to a species with a broad absorbance maximum centered at about 380 nm [O'Brien, D. F., Costa, L. F., & Ott, R. A. (1977) Biochemistry 16, 1295-1303]. On the basis of the results of a variety of chemical and spectroscopic tests, we show that this process corresponds to the production of free retinal plus opsin and not to the slow production of metarhodopsin II. Electron spin resonance studies using a novel disulfide spin-label that is covalently linked to rhodopsin indicate that the apparent arrest of the protein at the metarhodopsin I stage is not due to simple aggregation of the protein in this short-chain, saturated lipid bilayer but must be understood in terms of the effect of the lipid host on the conformational energies of individual protein molecules. Limited production of metarhodopsin II is observed under acidic conditions. Thus, the rhodopsin-dimyristoylphosphatidylcholine recombinants offer a unique system for the study of the effect of the phospholipid bilayer environment on the conformation of an intrinsic membrane protein.     

1H NMR spectroscopic studies of calcium-binding proteins. 3. Solution conformations of rat apo-alpha-parvalbumin and metal-bound rat alpha-parvalbumin

Lacking the extraordinary thermal stability of its metal-bound forms, apo-alpha-parvalbumin from rat muscle assumes two distinct conformations in aqueous solution. At 25 degrees C, its highly structured form predominates (Keq = 5.7; delta G degree = -4.3 kJ X mol-1); as deduced from both 1H NMR and circular dichroism (CD) spectroscopy, this conformation is exceedingly similar to those of its Mg(II)-, Ca(II)-, and Lu(III)-bound forms. The temperature dependences of several well-resolved aromatic and upfield-shifted methyl 1H NMR resonances and several CD bands indicate that the native, highly helical structure of rat apo-alpha-parvalbumin is unfolded by a concerted mechanism, showing no indication of partially structured intermediates. The melting temperature, TM, of rat apo-alpha-parvalbumin is 35 +/- 0.5 degrees C as calculated by both spectroscopic techniques. By 45 degrees C, rat apo-alpha-parvalbumin unfolds entirely, losing the tertiary structure that characterizes its folded form: not only are the ring-current-shifted aromatic and methyl 1H NMR resonances leveled, but the 262- and 269-nm CD bands are also severely reduced. As judged by the decrease in the negative ellipticity of the 222-nm CD band, this less-structured form of rat apo-alpha-parvalbumin shows an approximate 50% loss in apparent alpha-helical content compared to its folded state. Several changes in the 1H NMR spectrum of rat apo-alpha-parvalbumin were exceptionally informative probes of the specific conformational changes that accompany metal ion binding and metal ion exchange. In particular, the line intensities of the ortho proton resonance of Phe-47, the unassigned downfield-shifted alpha-CH resonances from the beta-sheet contacts between the metal-binding loops, the C2H resonance of His-48, and the epsilon-CH3 resonance of an unassigned Met residue were monitored as a function of added metal to determine the stability constants of several metal ion-parvalbumin complexes. We conclude that Mg(II) binds to the CD and EF sites independently, its affinity for the EF site being almost twice that for the CD site. Mg(II)----Ca(II) exchange showed that the CD-site Mg(II) is displaced first, in contrast to Lu(III)'s preferential displacement of the EF-site Ca(II) as determined from the Ca(II)----Lu(III) exchange experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro binding of synthetic acylated lipid-associating peptides to high-density lipoproteins: effect of hydrophobicity

To measure the effect of hydrophobicity on the binding of model apoproteins to lipoproteins, we synthesized a 15 amino acid lipid-associating peptide (LAP) with acyl chains of various lengths (0-18 carbons) bound to the N-terminal amino acid through a peptide bond. The acylated LAPs preferentially bound to high-density lipoprotein (HDL) and were activators of lecithin:cholesterol acyltransferase. Circular dichroic spectra indicated that the LAP association with phospholipid was accompanied by increased alpha-helical structure. The LAPs self-associated in solution as judged from tryptophan fluorescence analysis. These characteristics, which are comparable to those of apolipoprotein A-I, were strongly dependent upon the acyl chain length of the LAPs. The equilibrium constants (Keq) for the association of LAPs to reassembled HDL were measured by equilibrium dialysis at several temperatures. At 37 degrees C, Keq increased by 3 orders of magnitude as the number of carbon units was increased from 0 to 16; there was a log-linear relationship between Keq and the acyl chain length. The free energy of association (delta Ga) decreased by a constant value for each methylene unit added to the acyl chain (0.35 kcal mol-1), clearly demonstrating a strict hydrophobic effect. This change of delta Ga was enthalpy rather than entropy driven. Our data show that, with all other parameters including putative alpha-helicity, sequence, and molecular weight being constant, the binding of a lipid-associating peptide to lipoprotein is governed by its hydrophobicity.     

Rhodopsin photointermediates in two-dimensional crystals at physiological temperatures

Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystal suspensions were studied by measuring the time-dependent absorbance changes produced after excitation with 7 ns laser pulses at 15, 25, and 35 degrees C. The crystalline environment favored the Meta I(480) photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 degrees C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I(480) decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35 degrees C and quenched to 19 degrees C showed that a rapid equilibrium existed on the approximately 1 s time scale, which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII(a)) forms in 2D rhodopsin crystals but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.     

Binding studies of polypeptide hormones to bovine neurophysins.

Experimental binding isotherms of [9-glycinamide-1-(14)C]oxytocin and [9-glycinamide-1-(14)C]arginine vasopressin to purified neurophysins I and II at pH = 4.4, 5.4, 6.5, 7.4, and 8.5 and 6 degrees, 22 degrees, and 37 degrees in aqueous buffers are reported. For purposes of comparison, binding isotherms for [4-glycine-1-(14)C]oxytocin to neurophysin II and I in aqueous buffer, and [9-glycinamide-1-(14)C]oxytocin to neurophysin II in dimethylsulfoxide under selected conditions are also reported. A brief discussion of the interpretation of binding isotherms is entered into and apparent binding constants are derived. The results indicate that the interpretations presented in the literature up to now are much too simple. There are, in contrast, multiple binding sites of oxytocin and vasopressin to the neurophysins and large temperature dependences of the number of sites and their binding constants. We find, in fact, that at 37 degrees the binding of neurohypophysial hormones to the supposed storage proteins is rather weak even at the pH of maximum binding.     

Tautomeric Forms of Metarhodopsin

Light isomerizes the chromophore of rhodopsin, 11-cis retinal (formerly retinene), to the all-trans configuration. This introduces a succession of unstable intermediates—pre-lumirhodopsin, lumirhodopsin, metarhodopsin —in which all-trans retinal is still attached to the chromophoric site on opsin. Finally, retinal is hydrolyzed from opsin. The present experiments show that metarhodopsin exists in two tautomeric forms, metarhodopsins I and II, with λ_max 478 and 380 mµ. Metarhodopsin I appears first, then enters into equilibrium with metarhodopsin II. In this equilibrium, the proportion of metarhodopsin II is favored by higher temperature or pH, neutral salts, and glycerol. The change from metarhodopsin I to II involves the binding of a proton by a group with pK 6.4 (imidazole?), and a large increase of entropy. Metarhodopsin II has been confused earlier with the final mixture of all-trans retinal and opsin (λ_max 387 mµ), which it resembles in spectrum. These two products are, however, readily distinguished experimentally.