The dynamic properties of biological membranes.

Biological membranes must be viewed as highly dynamic, undergoing continuous structural fluctuations and changes in response to external perturbations. The study of liposomes by 31 P n.m.r. and fluorescence can reveal some of the motional characteristics of the different regions in a bilayer. Asymmetric lipid distribution and how this depends on the environment is also observed by n.m.r. The nature of the interaction of amine anaesthetics and of polypeptide antibodies with membranes is discussed in relation to their perturbing effect. The role of lipid mobility in modulating hormone-receptor interaction is discussed with reference to the binding of thyroid stimulating hormone.     

N.m.r. studies of metabolism in perfused organs

Several metabolites and intracellular pH in intact organs can be studied in a non-destructive manner by phorphorus nuclear magnetic resonance (31P n.m.r.). This possibility was demonstrated by us nearly five years ago. Since then we have developed the appropriate physiological techniques and improved the n.m.r. method for the study of animal hearts and kidneys. Here we described measurements aimed at clarifying three problesm. (1) Having measured the enzyme-catalysed fluxes between phosphocreatine and ATP by the method of saturation transfer n.m.r., we examine the relations between energy supply and heart rate in the isolated perfused rat heart. (2) We describe experiments to establish the validity of the perfusion model. For the first time, we report 31P n.m.r. measurements of an in vivo rat heart and compare the results with those obtained for the perfused rat heart. (3) Ischaemia and metabolism in rabbit kidneys is investigated to establish the relation between functional and metabolic recovery after a renal transplant operation.     

Magic angle sample spinning in inhomogeneously broadened biological systems

Recent advances in magic angle sample spinning experiments now permit observation of dilute spin high resolution n.m.r. spectra of arbitrary powder samples. In the 'slow-spinning' régime, for which the spinning rate is less than the size of the interaction that is being averaged, the spectra exhibit rotational side bands whose intensities contain information on chemical shift anisotropies. A technique for extracting shift anisotropies from side band intensities is discussed. Since many biologically interesting systems are solid or semi-solid in nature, this technique should find wide application to biological systems. Two illustrations of the point are given in this paper, namely, n.m.r. studies of membranes and of the phosphate-containing phases of bone.     

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluidity of intact erythrocyte membranes. Correction for fluorescence energy transfer from diphenylhexatriene to hemoglobin

Membranes of intact erythrocytes were labeled by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) using an improved labeling procedure described previously (Plásek, J. and Jarolím, P. (1987) Gen. Physiol. Biophys. 6, 425-437). The relationship between the steady-state DPH fluorescence anisotropy r and the mean corpuscular hemoglobin concentration (MCHC) was studied. Fluorescence anisotropy increased with increasing MCHC. A linear dependence of r = 0.0026 (MCHC) + 0.113 was obtained which enabled us to measure the fluidity of intact red cell membranes. Without this correction for fluorescence quenching by hemoglobin, incorrect conclusions about membrane fluidity could be made. This fact is demonstrated in a group of pyruvate kinase deficient patients compared with a group of healthy blood donors.     

Adrenocorticotropic hormone (ACTH)-lipid interactions. Implications for involvement of amphipathic helix formation

ACTH-lipid interactions were investigated by: (1) lipid-monolayer studies using several zwitterionic and anionic phospholipids and gangliosides, (2) permeability experiments by following the swelling rate of liposomes in isotonic glycerol solutions by light scattering, using liposomes of synthetic lipids and liposomes made of lipids extracted from light synaptic plasma membranes, and (3) by steady-state fluorescence anisotropy measurements on liposomes derived from light synaptic plasma membranes employing 1,6-diphenyl-1,3,5-hexatriene as fluorescent probe. (1) The monolayer experiments demonstrated an interaction with gangliosides GT1, GM1, dioleoylphosphatidic acid and phosphatidylserine, but little or no interaction with phosphatidylcholine or sphingomyelin. The interaction with monolayers of GT1 or phosphatidic acid decreased for ACTH1-13-NH2 and ACTH1-10. (2) The liposome experiments showed that 2 X 10(-5) M ACTH1-24 increased the glycerol permeability by 20% and decreased the activation energy only when liposomes derived from light synaptic plasma membranes were used. Treatment of the liposomes with neuraminidase abolished the ACTH-induced permeability increase. (3) Steady-state fluorescence depolarization measurements revealed that ACTH1-24, ACTH1-16-NH2 and ACTH1-10 did not change the fluidity of liposomes derived from light synaptic plasma membranes as sensed by diphenylhexatriene. It is concluded that ACTH1-24 can bind to negatively charged lipids and can form an amphipathic helix aligned parallel to the membrane surface involving the N-terminal residues 1 to 12, possibly to 16. Polysialogangliosides will favorably meet the condition of a high local surface charge density under physiological circumstances. It is suggested that ACTH-ganglioside interactions will participate in ACTH-receptor interactions.     

Scope and limitations of the simple 13C-n.m.r. method of structural analysis of carbohydrates: glucodisaccharides

The scope and limitations of the SIMPLE n.m.r. method (secondary isotope multiplet n.m.r. spectroscopy of partially labelled entities) has been investigated for a series of glucodisaccharies. ¹³C-SIMPLE n.m.r. measurements have been made on solutions of (1→1)- (α,α-trehalose), (1→2)- (sophorose and kojibiose), (1→3)- (laminaribiose), and (1→6)-linked (gentiobiose and isomaltose) glucodisaccharides in (CD₃)₂SO and the results combined with those previously published for (1→4)-linked analogues (maltose and cellobiose). Each linkage (and substitution) type gives rise to a unique pattern of ¹³C isotopomers which, in principle, may be used for complete assignment of the spectra and structural analysis of the molecule. The glucodisaccharides are difficult to analyse, compared with other disaccharides, because the presence of two glucose moieties leads to degeneracies of a few isotopic multiplets which cannot be differentiated by the magnitudes of the isotope effects. Assignments in aqueous solutions were obtained by using the DIS (differential isotope shift) n.m.r. method in conjunction with the results from SIMPLE n.m.r. In practice, nearly all of the signals can be assigned unequivocally and the remaining signals are choices between two possible assignments.     

Electrostatics of phosphoinositide bilayer membranes. Theoretical and experimental results.

We made fluorescence, electron paramagnetic resonance (EPR), electrophoretic mobility, and ionizing electrode measurements to study the effect of the monovalent lipid phosphatidylinositol (PI) and the trivalent lipid phosphatidylinositol 4,5-bisphosphate (PIP2) on the electrostatic potential adjacent to bilayer membranes. When the membranes were formed from mixtures of PI and the zwitterionic lipid phosphatidylcholine (PC), the Gouy-Chapman-Stern (GCS) theory described adequately the dependence of potential on distance (0, 1, 2 nm) from the membrane, mole % negative lipid, and [KCI]. Furthermore, all EPR and fluorescence probes reported identical surface potentials with a PC/PI membrane. With PC/PIP2 membranes, however, the anionic (coion) probes reported less negative potentials than the cationic (counterion) probes; the deviations from the GCS theory were greater for the coions than the counterions. Discreteness-of-charge theories based on the Poisson-Boltzmann equation incorrectly predict that deviations from the GCS theory should be greater for counterions than for coions. We discuss a consistent statistical mechanical theory that takes into account three effects ignored in the GCS theory: the finite size of the ions in the double layer, the electrical interaction between pairs of ions (correlation effects), and the mobile discrete nature of the surface charges. This theory correctly predicts that deviations from GCS theory should be negligible for monovalent lipids, significant for trivalent lipids, and greater for coions than for counterions.     

Measurement of protein rotational motion using frequency domain polarized fluorescence depletion.

Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are examined anaerobically in a fluorescence microscope. In time domain PFD experiments a several-microsecond pulse of linearly polarized light produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. Monitoring the decay of ground state depletion with a probe beam alternatively polarized, parallel, and perpendicular to the depletion pulse permits the triplet lifetime and rotational correlation time to be resolved and evaluated. We have now explored fluorescence depletion methods in the frequency domain to see whether such measurements could provide simpler and more efficient routine measurements of protein rotational relaxation than previous time domain PFD methods. An acousto-optic modulator (AOM) modulates the intensity of a 514.5 nm argon ion laser beam and a Pockels cell (PC) rotates its plane of polarization. These devices are driven by sinusoidal or square waves in fixed frequency relation, and rigidly phase locked, one to another. The fluorescence emitted from a sample then contains various overtones and combinations of the AOM and PC frequencies. The magnitude and phase of individual fluorescence signal frequencies are measured by a lock-in amplifier using a reference also phase-locked to both the AOM and PC. Specific frequencies permit evaluation of the rotational correlation time of the macromolecule and of the fluorophore triplet state lifetime, respectively. Measurement of bovine serum albumin rotation in glycerol solutions by this method is described.     

Progress in n.m.r. zeugmatography imaging

Applications of nuclear magnetic resonance (n.m.r.) zeugmatographic imaging to medical diagnosis and to medical, physiological, and biological research require the development of appropriate imaging instrumentation and ancillary techniques, as well as an understanding of the biological significance of the imaging results. A whole body imaging system, relying primarily upon reconstruction from projections, is under development in the expectation that the reconstruction approach will be the most practical one for many purposes. In addition, injectable magnetic reagents that can selectively change tissue water relaxation times and image contrast are under development so as to increase the specificity and versatility of the measurements. If very high magnetic fields are employed, 31P n.m.r. zeugmatography may be practical at very low resolution for human diagnostic studies and for experiments on perfused organs and small animals. Preliminary images, showing the spatial distributions of different phosphorus metabolites in the compartments of test objects, have been obtained at 146 MHz by reconstruction techniques.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing Laurdan generalized polarization

Organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors. The lipid composition of neuronal cells is unique and has been correlated with the increased complexity in the organization of the nervous system during evolution. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin_1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using the amphiphilic environment-sensitive fluorescent probe Laurdan. Our results show that the emission spectra of Laurdan display an additional red shifted peak as a function of increasing temperature in native as well as cholesterol-depleted membranes and liposomes made from lipid extracts of the native membrane. Interestingly, wavelength dependence of Laurdan generalized polarization (GP) in native membranes indicates the presence of an ordered gel-like phase at low temperatures, whereas characteristics of the liquid-ordered phase are observed at high temperatures. Similar experiments performed using cholesterol-depleted membranes show fluidization of the membrane with increasing cholesterol depletion. In addition, results from fluorescence polarization of DPH indicate that the hippocampal membrane is fairly ordered even at physiological temperature. The temperature dependence of Laurdan excitation GP provides a measure of the apparent thermal transition temperature and extent of cooperativity in these membranes. Analysis of time-resolved fluorescence measurements of Laurdan shows reduction in mean fluorescence lifetime with increasing temperature due to change in environmental polarity. These results constitute novel information on the dynamics of hippocampal membranes and its modulation by cholesterol depletion monitored using Laurdan fluorescence.     

The folding of an enzyme. V. H/2H exchange-nuclear magnetic resonance studies on the folding pathway of barnase: complementarity to and agreement with protein engineering studies.

Two major methods are currently being used to characterize transient intermediates during protein folding at the level of individual residues. Nuclear magnetic resonance (n.m.r.) measurements on the protection of peptide NH hydrogens against exchange with solvent during refolding can provide information about secondary structure formation. Protein engineering and kinetics can provide direct information about intramolecular interactions of protein side-chains and indirect evidence on secondary structure. These procedures have provided the most complete pictures so far about protein folding intermediates. Both methods have been applied to the characterization of an intermediate in the refolding of barnase. Although the two methods give complementary information, there are some regions of the protein where the methods overlap well. We show that, with one possible exception that is obscure, n.m.r. and protein engineering give identical results for those interactions that can be analysed by both methods. This suggests that these are valid approaches for the study of protein folding intermediates in the case of barnase and that the combination of the methods is a powerful analytical procedure. Information provided by n.m.r. data that is complementary to the protein engineering experiments is: (1) early formation of the C terminus of helix2; (2) early formation of helix3; (3) early formation of several beta-turns (46-49, 101-104 in loop5); and (5) partial formation of loop5. Confirmatory evidence of protein engineering data on the intermediate is: (1) helix1 is complete from residues 10 to 18; (2) the interactions between all beta-strands are present; (3) part of loop2 is not formed; (4) part of loop3 is formed; and (5) some specific tertiary interactions are not made. For some interactions the protein engineering and H/2H exchange methods overlap directly. The information obtained for direct overlap is self consistent.     

Electrogenic and nonelectrogenic ion fluxes across lipid and mitochondrial membranes mediated by monensin and monensin ethyl ester

Monensin is a carrier of cations through lipid membranes capable of exchanging sodium (potassium) cations for protons by an electroneutral mechanism, whereas its ethyl ester derivative ethyl-monensin is supposed to transport sodium (potassium) cations in an electrogenic manner. To elucidate mechanistic details of the ionophoric activity, ion fluxes mediated by monensin and ethyl-monensin were measured on planar bilayer lipid membranes, liposomes, and mitochondria. In particular, generation of membrane potential on liposomes was studied via the measurements of rhodamine 6G uptake by fluorescence correlation spectroscopy. In mitochondria, swelling experiments were expounded by the additional measurements of respiration, membrane potential, and matrix pH. It can be concluded that both monensin and ethyl-monensin can perform nonelectrogenic exchange of potassium (sodium) ions for protons and serve as electrogenic potassium ion carriers similar to valinomycin. The results obtained are in line with the predictions based on the crystal structures of the monensin complexes with sodium ions and protons (Huczyński et al., Biochim. Biophys. Acta, 1818 (2012) pp. 2108–2119). The functional activity observed for artificial membranes and mitochondria can be applied to explain the activity of ionophores in living systems. It can also be important for studying the antitumor activity of monensin.     

A master equation theory of fluorescence induction, photochemical yield, and singlet-triplet exciton quenching in photosynthetic systems.

A master equation theory is formulated to describe the dependence of the fluorescence yield (phi) in photosynthetic systems on the number of photons (Y) absorbed per photosynthetic unit (or domain). This theory is applied to the calculation of the dependence of the fluorescence yield on Y in (a) fluorescence induction, and (b) singlet exciton-triplet excited-state quenching experiments. In both cases, the fluorescence yield depends on the number of previously absorbed photons per domain, and thus evolves in a nonlinear manner with increasing Y. In case a, excitons transform the photosynthetic reaction centers from a quenching state to a nonquenching state, or a lower efficiency of quenching state; subsequently, absorbed photons have a higher probability of decaying by radiative pathways and phi increases as Y increases. In case b, ground-state carotenoid molecules are converted to long-lived triplet excited-state quenchers, and phi decreases as Y increases. It is shown that both types of processes are formally described by the same theoretical equations that relate phi to Y. The calculated phi (Y) curves depend on two parameters m and R, where m is the number of reaction centers (or ground-state carotenoid molecules that can be converted to triplets), and R is the ratio phi (Y leads to infinity)/(Y leads to 0). The finiteness of the photosynthetic units is thus taken into account. The m = 1 case corresponds to the "puddle" model, and m leads to infinity to the "lake," or matrix, model. It is shown that the experimental phi (Y) curves for both fluorescence induction and singlet-triplet exciton quenching experiments are better described by the m leads to infinity cases than the m = 1 case.     

Steady-state fluorescence polarization data in membranes. Resolution into physical parameters by an extended Perrin equation for restricted rotation of fluorophores

An extended Perrin equation is derived applicable to the restricted rotation of fluorophores. The equation results in a relation between time-resolved (r infinity) and steady-state fluorescence anisotropy (rs) data. This relation contains a parameter m, which expresses the difference between rotational diffusion in a lipid membrane and that in an isotropic reference oil having the same rs value. The relation is in agreement with rs, r infinity literature data for a variety of artificial and biological membranes labeled with various probes. Cholesterol and fatty acyl unsaturation affect the value of m, but temperature does not. The results indicate that, as far as fluorescence depolarization is concerned, either liposomes of saturated phospholipids without cholesterol or liposomes of unsaturated phospholipids containing cholesterol are good model systems for biological membranes. The accuracy of estimating order parameters or rotational diffusion constants from rs data is discussed. The formalism described here introduces a novel way of applying Arrhenius plots and allows for an unambiguous interpretation of rs data.     

Telomere length measurements using digital fluorescence microscopy.

BACKGROUND: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.     

Dual-wavelength ratiometric fluorescence measurements of membrane potential

This work shows that the voltage across membranes in two very different preparations, lipid vesicles in suspension and individual HeLa cells under a microscope, is linearly related to the ratio of fluorescence excited from the two wings of the absorption spectrum of a voltage-sensitive dye. The dye di-4-ANEPPS [1-(3-sulfonatopropyl)-4-[beta-[2-(di-n-butylamino)-6-naphthyl] vin yl]pyridinium betaine] is well characterized from earlier investigations and responds via a rapid (less than millisecond) spectral shift to membrane potential changes. The resultant small change in fluorescence intensity monitored at a single wavelength is useful for measurements of temporally well-defined voltage transients such as action potentials. The dual-wavelength approach described in this work extends the usefulness of this fast potentiometric dye by filtering out complex or artifactual changes in fluorescence intensity and providing a voltage-dependent signal that is internally standardized. Thus, rapid measurements of membrane potential are made possible in nonexcitable cells.     

An evaluation of the combined use of nuclear magnetic resonance and distance geometry for the determination of protein conformations in solution

An evaluation of the potential of nuclear magnetic resonance (n.m.r.) as a means of determining polypeptide conformation in solution is performed with the aid of a new distance geometry program which is capable of computing complete spatial structures for small proteins from n.m.r. data. Ten sets of geometric constraints which simulate the results available from n.m.r. experiments of varying precision and completeness were extracted from the crystal structure of the basic pancreatic trypsin inhibitor, and conformers consistent with these constraints were computed. Comparison of these computed structures with each other and with the original crystal structure shows that it is possible to determine the global conformation of a polypeptide chain from the distance constraints which are available from n.m.r. experiments. The results obtained with the different data sets also provide a standard by which the quality of protein structures computed from n.m.r. data can be evaluated when no crystal structure is available, and indicate directions in which n.m.r. experiments for protein structure determination could be further improved.     

Methods to measure the lateral diffusion of membrane lipids and proteins

In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are presented in that order. FRAP and FCS methodologies are described for a dedicated wide field microscope although many confocal microscopes now have software permitting these measurement to be made; nevertheless, the principles of the measurement are the same for a wide field or confocal microscope. SPT can be applied to trace the movements of single fluorescent molecules in membranes but this aspect will not be treated in detail.     

Practical guidelines for dual-color fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS on a commercially available setup. Although FCCS is closely related to two-color confocal microscopy, critical adjustments and test measurements are necessary to establish successful FCCS measurements, which are described in a step-by-step manner. Moreover, we discuss control experiments for a negative cross-correlation artifact, arising from a lack of detection volume overlap, and a positive artifact, arising from cross-talk. FCCS has been applied to follow molecular interactions in solutions, on membranes and in cells and to analyze dynamic colocalization during intracellular transport. It is a technique that is expected to see new applications in various fields of biochemical and cell biological research.