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Biomedical subjects

L Stryer

Publications and source records attributed to L Stryer.

At least 109 records · Page 6Linked to original sources
PubMed1977

Accessibility of the carbohydrate moiety of membrane-boound rhodopsin to enzymatic and chemical modification.

Galactose was specifically inserted into the carbohydrate moiety of rhodopsin by incubating retinal disk membranes with UDP-galactose: N-acetylglucosamine galactosyltransferase. The stoichiometry of labeling ranged from 1.2 to 1.8 (average = 1.5) residues of galactose per molecule of rhodopsin, indicating that some or all of the oligosaccharide chains of membrane-bound rhodopsin are readily accessible to enzymatic modification. These modified membranes were treated with galactose oxidase to generate an aldehyde at the C-6 position of the inserted galactose units. The enzymatically-oxidized membranes were then reacted with dansyl hydrazide to yield a fluorescent hydrazone which is sufficiently stable to permit spectroscopic analysis. This procedure for the specific attachment of a spectroscopic probe should be applicable to a wide variety of membrane glycoproteins.

Animals↗
PubMed1976

Rapid-flow resonance Raman spectroscopy of photolabile molecules: rhodopsin and isorhodopsin.

We have devised a method for obtaining the resonance Raman spectrum of a photolabile molecule before it is modified by light. The essence of this technique is that the sample is flowed through the light beam at a sufficiently high velocity so that the fraction of photoisomerized (or photodestroyed) molecules in the illuminated volume is very low. This rapid-flow technique has enabled us to measure the resonance Raman spectrum of unphotolyzed bovine rhodopsin in Ammonyx LO detergent solution and in sonicated retinal disc membranes. The major features of these spectra, which are very similar to one another, are the protonated Schiff base line near 1660 cm-1, the ethylenic line at 1545 cm-1, lines due to skeletal modes at 1216, 1240, and 1270 cm-1, and a line due to C-H bending at 971 cm-1. The resonance Raman spectrum of unphotolyzed isorhodopsin formed by the addition of 9-cis-retinal to opsin was also measured. The spectrum of isorhodopsin is more complex and differs markedly from that of rhodopsin. In isorhodopsin, the ethylenic line is shifted to 1550 cm-1, and there are six lines between 1153 and 1318 cm-1. The rapid-flow technique described here makes it feasible to control the extent of interaction between light and any photolabile molecule. We present a theory for predicting the effective sample composition in the illuminated volume as a function of the flow rate, light intensity, and spectral characteristics of the photolabile species.

Animals↗
PubMed1976

Retinal has a highly dipolar vertically excited singlet state: implications for vision.

We have measured the effect of an intense electric field on the absorption spectrum of solutions of all-trans retinal, its unprotonated Schiff base with n-butylamine, and the Cl- salt of this protonated Schiff base. The field-induced change in extinction coefficient as a function of wavelength was analyzed to determine the ground-state dipole moment (mug), the change in dipole moment on excitation (deltamu), and the direction of mug and deltamu). These experiments have shown that all three species become highly dipolar upon excitation to the first allowed excited singlet state (deltamu = 15.6, 9.9, 12D, respectively). The ground-state and excited-state dipole moments are nearly parallel to the long axis of these molecules. Excitation is accompanied by a shift of negative charge toward the carbonyl or Schiff base terminus, making the ionone end of these molecules positively charged. The large excited state dipole moment of all-trans retinal indicates that the vertically excited state, which is of 1Bu parentage (C2h), has become significantly mixed with even-parity states. On the basis of previous theoretical calculations, this mixing is expected to facilitate isomerization in the singlet manifold. We have also found that 11-cis retinal has a large deltamu (12.7 +/- 1.4 D) on excitation. In the visual pigments, the interaction of the excited-state dipole moment of retinal with a suitably located charged group could control the position of the absorption maximum. Also, the large shift in charge density upon excitation of retinal may lead to new electrostatic interactions between the chromophore and the protein that would act as a driving force for the initial conformational changes in visual excitation.

Chemical Phenomena↗
PubMed1972

Proximity relationships in rhodopsin.

Energy transfer was used as a spectroscopic ruler to deduce proximity relationships within bovine rhodopsin in digitonin solution. Rhodopsin was specifically labeled with fluorescent chromophores at three sites. Site A was alkylated by fluorescent derivatives of iodoacetamide. Site B was labeled by fluorescent disulfides, by a disulfide-sulfhydryl interchange reaction. Sites A and B are sulfhydryl residues. Acridine derivatives were tightly bound to site C by noncovalent interactions. The labeled rhodopsins retained their 500-nm absorption band and were regenerable after bleaching, suggesting that the fluorescent probes did not grossly perturb the conformation of the protein. A fluorescent chromophore at one of these sites served as the energy donor, while 11-cis retinal was the energy acceptor. The efficiency of singlet-singlet energy transfer was determined from the quantum yield and excited-state lifetime of the donor in the presence and absence of the acceptor. By Förster's theory, the apparent distances between 11-cis retinal and sites A, B, and C were calculated to be 75,55, and 48 A, respectively. Energy transfer measurements on rhodopsin labeled at two of these sites gave these apparent distances: 35 A for A to B, 32 A for A to C, and 30 A for B to C. These energy transfer studies suggest that the rhodopsin molecule has a length of at least 75 A. Thus, the rhodopsin molecule appears to be sufficiently long to traverse the disc membrane. Rhodopsin might act as a light-controlled gate.

Acridines↗
PubMed1971

Fluorescence spectroscopy of an oriented model membrane.

We have devised a simple method that makes it feasible to apply fluorescence techniques to lipid bilayer membranes to elucidate aspects of their structure and dynamics. Fluorescence excitation, emission, and polarization spectra were obtained from a single spherical bilayer membrane consisting of oxidized cholesterol and fluorescent probe. The emission transition moments of N,N'-di(octadecyl)oxacarbocyanine and 12-(9-anthroyl)-stearic acid were found to be aligned parallel to the plane of the bilayer, whereas that of p-bis-[2-(4-methyl-5-phenyloxazolyl)]-benzene was aligned in a perpendicular direction. All three probes exhibited appreciable rotational mobility, parallel to the plane of the bilayer, in durations of nanoseconds. An attractive feature of this model membrane is that fluorescence measurements can be made at the same time as electrical measurements and perturbations. Also, it may be possible to incorporate functional protein assemblies into this model and to use fluorescence spectroscopy to delineate some aspects of their assembly and function.

Cholesterol↗
PubMed1970

Fluorescent probes of biological membranes.

FLUORESCENT AND PHOSPHORESCENT PROBES THAT HAVE READILY INTERPRETABLE EMISSION PROPERTIES CAN BE SPECIFICALLY INSERTED INTO BIOLOGICAL MACROMOLECULES TO REVEAL FACETS OF THEIR STRUCTURE AND DYNAMICS: (1) Proximity. Singlet-singlet and triplet-singlet energy transfer can serve as spectroscopic rulers in the 10-65 A range, whereas triplet-triplet transfer can be used to show that two groups are less than about 12 A apart. (2) Rotational mobility. Nanosecond fluorescence polarization measurements can reveal whether a macromolecular system has any modes of flexibility in times of nanoseconds. (3) Polarity. The presence of mobile dipoles in the environment of certain chromophores is reflected in their fluorescence quantum yield and emission spectrum. We have synthesized a number of new fluorescent probes for biological membranes. Anthroyl stearic acid (I), dansyl phosphatidyl ethanolamine (II), and octadecyl naphthylamine sulfonic acid (III) are readily incorporated into bilayer vesicles composed of phosphatidyl choline. The emission spectra of these probes in the vesicles indicate that the chromophore of I is located in the hydrocarbon region, that of II is located in the glycerol layer, and that of III is located at the aqueous interface of the bilayer. Thus, fluorescent chromophores can be selectively placed in different transverse regions of a model membrane system.

Anthracenes↗