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A M Kleinfeld

Publications and source records attributed to A M Kleinfeld.

47 records · Page 3Linked to original sources
PubMed1985

Tryptophan imaging of membrane proteins.

A theoretical analysis of resonance energy transfer between protein tryptophan and the n-(9-anthroyloxy) (AO) fatty acid probes has been carried out to evaluate its potential use in determining the tryptophan distribution in membrane proteins. The Förster theory for two-dimensional energy transfer was formulated to calculate multiple donor (tryptophan) transfer efficiencies to ensembles of AO probes at different depths in the bilayer. The variation of transfer efficiency with AO probe depth is found to be a sensitive function of tryptophan position and the protein radius but not the dipole-dipole orientation factor or the decay heterogeneity of the donor. For single tryptophan-containing proteins the model predicts that the tryptophan position can be determined with a precision of about 2 A. Although for multiple tryptophans there is appreciable deterioration in resolution, it is still possible to determine the essential features of the distribution such as its first two moments. The positions determined by this method are the projections of the tryptophan positions on a plane perpendicular to the membrane surface, since the probes distribute uniformly around the protein. To analyze the data, a Monte Carlo approach has been developed to search for tryptophan distributions compatible with the observed efficiencies and to display the results in terms of a tryptophan density map. It is shown that even for cases in which little is known about the quantum yield distribution, significant information can be determined about the tryptophan spatial distribution.

Animals↗
PubMed1985

Energy-transfer study of cytochrome b5 using the anthroyloxy fatty acid membrane probes.

Resonance energy transfer was used to study the structure of cytochrome b5 and its nonpolar segment reconstituted into sonicated vesicles of dimyristoylphosphatidylcholine. The n-(9-anthroyloxy) (AO) fatty acid probes were added to these vesicles, and energy-transfer measurements were carried out between tryptophan and AO, tryptophan and the heme moiety of cytochrome b5, and AO and heme. Results of these measurements were analyzed by using the methods outlined in the previous paper [Kleinfeld, A. M. (1985) Biochemistry (preceding paper in this issue)]. We find, in agreement with Fleming et al. [Fleming, P. J., Koppel, D. E., Lau, A. L. Y., & Strittmatter, P. (1979) Biochemistry 18, 5458-5464], that the fluorescent tryptophan in both forms of the protein is buried about 20 A from the surface and that most of the fluorescence is associated with a single tryptophan. The results are consistent with the AO probe distance of closest approach to the protein, greater for whole b5 than for the nonpolar peptide. The tryptophan-heme and AO-heme measurements indicate that the heme moiety is about 15 A from the surface of the membrane. The agreement of our results with the previous studies supports the description of tryptophan-AO energy transfer outlined in the preceding paper.

Animals↗
PubMed1984

Hairpin configuration of H-2Kk in liposomes formed by detergent dialysis.

H-2Kk is a transmembrane glycoprotein having the N-terminal region of the heavy chain exposed at the cell surface and the C-terminal region exposed at the cytoplasmic face in its native configuration in the plasma membrane. The configuration of H-2Kk in liposomes formed by detergent dialysis was investigated by using fluorescently labeled H-2 and Co2+ ions to quench fluorescence. H-2Kk was incorporated into sealed lipid vesicles when deoxycholate was removed by dialysis from a mixture of protein and lipid. Including 20 mM carboxyfluorescein (CF) in the mixture prior to dialysis resulted in CF trapped inside the vesicles at concentrations where self-quenching occurred. Vesicles with CF trapped inside were shown to be osmotically active and impermeable to Na+ and Co2+ ions. In order to examine the configuration of H-2Kk in these liposomes, the heavy chain was covalently labeled by using the sulfhydryl reactive fluorescent reagents fluorescein-5-ylmaleimide (NFM) or 5-[[2-[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS). In both cases, approximately equal amounts of fluorescent label were incorporated into the N- and C-terminal regions of the protein. Incorporation of the labeled H-2 into liposomes and examination of the effect of Co2+ on the fluorescence showed that all of the label was accessible to quenching by Co2+ and thus exposed on the outside of the liposome. The results demonstrate that the H-2Kk is incorporated into these liposomes in a hairpin configuration, not in the transmembrane configuration found in native membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Partition of fatty acids and fluorescent fatty acids into membranes.

We have measured the membrane/water partition of free fatty acids, of a fluorescent analogue, and of chlorpromazine into plasma membranes from lymphoma, platelets, red cells, and liposomes of egg phosphatidylcholine. Three different methods were used: hygroscopic desorption [ Conrad , M.J., & Singer, S.J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5202-5206; Conrad , M.J., & Singer, S.J. (1981) Biochemistry 20, 808-818], centrifugation, and fluorescence titration. The partition coefficients for chlorpromazine and all fatty acid/membrane combinations were in the range 10(2)-10(5). No significant partition difference was observed between liposomes and plasma membranes. These results suggest, therefore, that fatty acids and chlorpromazine can readily partition into artificial and cell plasma membranes.

Animals↗
PubMed1981

The lack of relationship between fluorescence polarization and lateral diffusion in biological membranes.

An investigation has been carried out of the relationship between changes in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and concomitant changes in the lateral diffusion of proteins and lipid probes in membranes. Plasma membranes from lymphocytes and a CH1 mouse lymphoma line were treated with up to 70 mol% (relative to the total membrane phospholipid) of oleic or linoleic fatty acids. Under these conditions the fluorescence polarization of DPH decreased by between 8 and 15% which, in the framework of the microviscosity approach, suggests a membrane fluidity change of between 20 and 50%. The lateral diffusion coefficients of surface immunoglobin and the lipid probes 3,3'-dioctadecylindocarbocyanine and pyrene were also measured in these membranes using the fluorescence photobleaching recovery technique and the rate of pyrene excimer formation. The diffusion rates were found to be unaffected by the presence of free fatty acids. Hence despite large 'microviscosity' changes as reported by depolarization of DPH fluorescence, lateral diffusion coefficients are essentially unchanged. This finding is consistent with the idea that perturbing agents such as free fatty acids do not cause a general fluidization of the membrane but act locally to alter, for example, protein function. It is also consistent with the suggestion that lateral mobility of membrane proteins is not modulated by the lipid viscosity.

Animals↗
PubMed1981

Emission-wavelength-dependent decay of the fluorescent probe N-phenyl-1-naphthylamine.

We have measured the fluorescence decay of N-phenyl-1-naphthylamine using the phase-modulation method, in several solvent systems and egg phosphatidylcholine vesicles. The decay is monoexponential in pure solvents (both polar and non-polar) of low viscosity. In polar viscous solvents or in non-polar solvents containing an added polar solute, the decay is heterogeneous and emission wavelength dependent. In such cases, dielectric relaxation and/or excited-rate complexing give rise to a shift of the emission spectrum on the nanosecond time scale. Emission-wavelength-dependent decay was also observed when N-phenyl-1-naphthylamine was bound to egg phosphatidylcholine vesicles. From these results as well as the position of the emission spectral maximum, we conclude that N-phenyl-1-naphthylamine probes the ester-carbonyl region of the phospholipid acyl chains, where it undergoes an excited-state reaction. This result contradicts the often made assumption that N-phenyl-1-naphthylamine probes the deeper hydrocarbon region of the bilayer.

1-Naphthylamine↗
PubMed1981

Emission wavelength-dependent decay of the 9-anthroyloxy-fatty acid membrane probes.

Using the phase-modulation technique, we have measured the fluorescence decay of 2- and 12-(9-anthroyloxy)-stearic acid (2- and 12-AS) and 16-(9-anthroyloxy)-palmitic acid (16-AP) bound to egg phosphatidylcholine vesicles or dissolved in nonpolar solvents. Heterogeneity analysis demonstrates that the decay is generally not monoexponential and exhibits large component variations across it emission spectrum. The mean decay time increases (and in parallel, the steady-state polarization decreases) monotonically with increasing wavelength from values at the blue end. The decay at the red side of the emission spectrum contains an exponential term with a negative amplitude, indicating that emission occurs from intermediates created in the excited-state. This behavior is interpreted as arising from intramolecular fluorophore relaxation occurring on the time scale of the fluorescence lifetime. We believe this to be the first study of wavelength-dependent fluorescent emission which is dominated by an intramolecular relaxation process. Although the three probes exhibit qualitatively similar effects, the emission band variations are greatest for 2-AS and smallest for 16-AP. The differences among the probes are not entirely due to environmental factors as demonstrated, for example, by the emission polarization differences observed in the isotropic solvent paraffin oil. In summary, while these findings point out some of the complexities in the 9-anthroyloxy-fatty acids as membrane probes, they also indicate how these complexities might be used as a sensitive measure of lipid-probe interaction.

Anthracenes↗