Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction.

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

Model-independent analysis of the orientation of fluorescent probes with restricted mobility in muscle fibers.

The orientation of proteins in ordered biological samples can be investigated using steady-state polarized fluorescence from probes conjugated to the protein. A general limitation of this approach is that the probes typically exhibit rapid orientational motion ("wobble") with respect to the protein backbone. Here we present a method for characterizing the extent of this wobble and for removing its effects from the available information about the static orientational distribution of the probes. The analysis depends on four assumptions: 1) the probe wobble is fast compared with the nanosecond time scale of its excited-state decay; 2) the orientational distributions of the absorption and emission transition dipole moments are cylindrically symmetrical about a common axis c fixed in the protein; 3) protein motions are negligible during the excited-state decay; 4) the distribution of c is cylindrically symmetrical about the director of the experimental sample. In a muscle fiber, the director is the fiber axis, F. All of the information on the orientational order of the probe that is available from measurements of linearly polarized fluorescence is contained in five independent polarized fluorescence intensities measured with excitation and emission polarizers parallel or perpendicular to F and with the propagation axis of the detected fluorescence parallel or perpendicular to that of the excitation. The analysis then yields the average second-rank and fourth-rank order parameters ( and ) of the angular distribution of c relative to F, and and , the average second-rank order parameters of the angular distribution for wobble of the absorption and emission transition dipole moments relative to c. The method can also be applied to other cylindrically ordered systems such as oriented lipid bilayer membranes and to processes slower than fluorescence that may be observed using longer-lived optically excited states.

Major histocompatibility complex class I protein conformation altered by transmembrane potential changes.

The nature of charge distributions in membrane-bound macromolecular structures renders them susceptible to interaction with transmembrane potential fields. As a result, conformational changes in such species may be expected to occur when this potential is altered. We have detected reversible conformational change in the major histocompatibility complex (MHC) class I antigen in the plasma membrane of human JY cells, as monitored by flow-cytometric resonance energy-transfer, upon reduction of the transmembrane potential (depolarization). This change increased the intramolecular energy-transfer efficiency between fluorescent donor- and acceptor-labeled monoclonal antibodies directed, respectively, to epitopes on the light (beta 2-microglobulin) and the heavy chains of the MHC class I antigen. Repolarization of the depolarized samples restored the energy-transfer efficiency to the original values measured before depolarization. Depolarization caused similar relative changes in fluorescence resonance energy-transfer efficiency when Fab fragments were used for labeling MHC class I complex, suggesting that the observed phenomenon is not restricted to whole monoclonal antibodies.

Role of interleukin-3 in the regulation of intracellular K+ homeostasis in cultured murine haemopoietic cells.

Comparative fluorimetric, flow cytofluorimetric and fluorescence ratiometric determinations of intracellular K+ concentrations in murine haemopoietic cells (FDCP-Mix clone A4) cultured in the presence and absence of the specific growth factor Interleukin-3 were carried out with the fluorescent potassium-binding benzofuran-isophthalate acetoxymethyl ester probe. Cell suspensions kept in the absence of Interleukin-3 for 5 hours exhibited lower fluorescence intensities and smaller fluorescence ratios than their growth-factor-replete counterparts, an effect found to be reversible by readdition of the growth factor. It is concluded that Interleukin-3 deprivation of these cells leads to loss of intracellular K+. It is tentatively suggested that this deprivation-induced K+ loss might be associated with an early event in apoptotic cell death.

Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes.

The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.

Interleukin-3-specific modification of cell membrane "fluidity" of haemopoietic cells.

The work reported here clearly demonstrates that a specific growth factor, interleukin-3 (IL-3), which acts on multipotent haemopoietic stem cells as well as on committed myeloid progenitor cells of different lineages (Schrader, 1988; Whetton and Dexter, 1986), specifically induces a modification of the physical state ("fluidity") of the cell membranes of two IL-3-responsive and apparently normal haemopoietic cell lines. Furthermore, in a derived IL-3 independent myeloid leukaemic cell line, no such physical response to IL-3 binding was observed. The rapidity of the "normal" response suggests further that it may be associated with, or even constitute per se a critical early effect elicited by IL-3 in sensitive cells, the necessity for which is abrogated in the malignant derivative.

Interpretation of fluorescence photobleaching recovery experiments on oriented cell membranes.

Both quantitative and qualitative aspects of the interpretation of fluorescence photobleaching recovery experiments as typically practised to obtain information on lateral diffusion processes in cell membranes are called into question in view of the polarized nature of the laser light sources routinely employed. Protocols which will eliminate the effects elicited under these conditions by any concurrent slow rotational diffusion are delineated.

Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in model lipid bilayer membrane systems by resonance excitation energy transfer.

A fluorescent phospholipid derivative, the fluoresceinthiocarbamyl adduct of a natural phosphatidylethanolamine, has been synthesized and incorporated into sonicated single-bilayer vesicles of egg lecithin and dipalmitoyllecithin. The surface location of this probe has been confirmed by using extrinsic fluorescence quenching studies together with steady-state emission anisotropy measurements. Electronic excitation energy transfer between 1,6-diphenyl-1,3,5-hexatriene incorporated within the hydrophobic core of the bilayer and the novel derivative has been investigated to estimate the depth within the bilayer at which the former is located. Efficiencies have been measured for two different phospholipids, egg lecithin and dipalmitoyllecithin, in the latter case both above and below the phospholipid phase transition, with and without added cholesterol. The observed dependence of the transfer efficiency on the acceptor concentration was compared with that calculated according to Förster theory applied to random two-dimensional distributions of donor and acceptor molecules in parallel planes for various interplanar separations, taking into account orientational effects. The Förster R0 of about 45 A for this donor-acceptor pair is particularly well suited to such studies since it is of the order of the width of the bilayer. The experiments showed that energy-transfer spectroscopy can provide useful quantitative information as to the transverse location of diphenylhexatriene in homogeneous phospholipid bilayers and may also reflect lateral partitioning of donor or of both donor and acceptor into different phases in systems exhibiting phase separations.

Rotational diffusion of calcium-dependent adenosine-5'-triphosphatase in sarcoplasmic reticulum: a detailed study.

The Ca2+-Mg2+ adenosine-5'-triphosphatase (ATPase) in sarcoplasmic reticulum has been covalently labeled with the phosphorescent triplet probe erythrosinyl 5-isothiocyanate. The rotational diffusion of the protein in the membrane at 25 degrees C was examined by measuring the time dependence of the phosphorescence emission anisotropy. Detailed analysis of both the total emission S(t) = Iv(t) + 2IH(t) and anisotropy R(t) = [Iv(t) - IH(t)]/[Iv(t) + 2IH(t)] curves shows the presence of multiple components. The latter is incompatible with a simple model of protein movement. The experimental data are consistent with a model in which the sum of four exponential components defines the phosphorescence decay. The anisotropy decay corresponds to a model in which the phosphor itself or a small phosphor-bearing segment reorients on a sub-microsecond time scale about an axis attached to a larger segment, which in turn reorients on a time scale of a few microseconds about an axis fixed in the frame of the ATPase. A fraction of the protein molecules rotate on a time scale of 100-200 microseconds about the normal to the bilayer, while the rest are rotationally stationary, at least on a sub-millisecond time scale.

The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer.

The measurement of the efficiency of Förster long-range resonance energy transfer between donor (D) and acceptor (A) luminophores attached to the same macromolecular substrate can be used to estimate the D-A separation, R. If the D and A transition dipoles sample all orientations with respect to the substrate (the isotropic condition) in a time short compared with the transfer time (the dynamic averaging condition), the average orientation factor less than K2 greater than is 2/3. If the isotropic condition is not satisfied but the dynamic averaging condition is, upper and lower bounds for less than K2 greater than, and thus R, may be obtained from observed D and A depolarizations, and these limits may be further narrowed if the transfer depolarization is also known. This paper offers experimental protocols for obtaining this reorientational information and presents contour plots of less than K2 greater than min and less than K2 greater than max as functions of generally observable depolarizations. This permits an uncertainty to be assigned to the determined value of R. The details of the D and A reoreintational process need not be known, but the orientational distributions are assumed to have at least approximate axial symmetry with respect to a stationary substrate. Average depolarization factors are derived for various orientational distribution functions that demonstrate the effects of various mechanisms for reorientation of the luminophores. It is shown that in general the static averaging regime does not lend itself to determinations of R.

Rotational relaxation of the "microviscosity" probe diphenylhexatriene in paraffin oil and egg lecithin vesicles.

The rotational relaxation of the widely used "microviscosity" probe, 1,6-diphenyl-1,3,5-hexatriene, was examined by the technique of nanosecond time-resolved fluorescence depolarization. The decays of the emission anisotropy were determined at five temperatures in the range 3-31 degrees both in a reference paraffin oil and in sonicated egg lecithin vesicles. These decays were complex in both media. Marked qualitative as well as quantitative differences were observed in the rotational behavior of the probe in the complex bilayer medium as opposed to the homogeneous reference solvent. The results are discussed in relation to the structure of the hydrophobic bilayer membrane interior and the concept of its "microviscosity".

Nanosecond time-dependent fluorescence depolarization of diphenylhexatriene in dimyristoyllecithin vesicles and the determination of "microviscosity".

The nanosecond time dependence of the fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene in L-alpha-dimyristoyllecithin vesicles was determined at temperatures above and below the midpoint of the gel-liquid crystalline transition. In neither case could the decay of the total fluorescent emission or the decay of the emission anisotropy be described adequately in terms of single exponential decay laws. At the lower temperature, the emission anisotropy did not approach zero in the time window available for measurement, a finding which may indicate that the range over which rotation of the probe can freely occur is restricted. The results are discussed in relation to the concept of microviscosity of bilayer membranes.

The interaction of the polyene antibiotic lucensomycin with cholesterol in erythrocyte membranes and in model systems. III. Characterization of spectral parameters.

The variations of optical density and fluorescence of lucensomycin are good indices of the binding of this polyenic antibiotic to membranes. The former parameter reflects more generally the binding to any site present in the membrane, while the latter is more specific for binding to cholesterol. The chromophore of the lucensomycin-cholesterol complex has a relatively long lifetime, is almost immobile in the membrane, and is not accessible to water-soluble fluorescence-quenching agents. The stoichiometry, evaluated fluorometrically, corresponds to about two cholesterol molecules per polyene. In colloidal cholesterol suspensions, the extent of binding as a function of free polyene concentration is described by rectangular hyperbolae, the dissociation constant being, however, dependent on the sterol concentration. In erythrocyte membranes, on the other hand, and even more markedly in model systems containing appropriate solvents, the combination between lucensomycin and the sterol sites is described by sigmoid titration curves, indicative of cooperative effects, and probably due to solvation of cholesterol.

Intramolecular energy transfer and molecular conformation.

A discussion of the range of applicability of Forster long-range energy transfer in the determination of macromolecular dimensions and conformational dynamics is given. Emphasis is laid on the effect of restrictions in the orientational freedom of donor and acceptor and on the importance of the orientational averaging regime. The usefulness and limitations of polarized emission measurements in this regard are discussed.

Isolation and spectral characterization of phycobiliproteins.

Several phycobiliproteins were prepared chromatographically pure and their absorption, fluorescence-emission, fluorescence-excitation and fluorescence-excitation polarization spectra determined. Changes in these spectra with ionic strength of the aqueous medium and chromoprotein concentration were interpreted in terms of interchromophore energy transfer and protein subunit equilibria. The complexity of the polarization spectra confirms the presence of different types of chromophore, designated sensitizing (;s') and fluorescing (;f'), in a single protein.