Theory and application of fluorescence homotransfer to melittin oligomerization.

Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.     

Determination of the orientational distribution and orientation factor for transfer between membrane-bound fluorophores using a confocal microscope

Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound fluorophores. We demonstrate how information about the orientation of fluorophores can be used to calculate the orientation factor kappa(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.     

Different effects of lipid chain length on the two sides of a membrane and the lipid annulus of MscL

Quenching of the fluorescence of Trp residues in a membrane protein by lipids with bromine-containing fatty acyl chains provides a powerful technique for measuring lipid-protein binding constants. Single Trp residues have been placed on the periplasmic and cytoplasmic sides of the mechanosensitive channel of large conductance MscL from Mycobacterium tuberculosis to measure, separately, lipid binding constants on the two faces of MscL. The chain-length dependence of lipid binding was found to be different on the two sides of MscL, the chain-length dependence being more marked on the cytoplasmic than on the periplasmic side. To determine if lipid binding constants are affected by the properties of the lipid molecules not in direct contact with MscL (the bulk lipid), the amount of bulk lipid present in the system was varied. The binding constant of the short-chain phospholipid didodecylphosphatidylcholine was found to be independent of the molar ratio of lipid/MscL pentamer over the range 500:1-50:1, suggesting that lipid binding constants are determined largely by the properties of the lipid molecules interacting directly with MscL. These results point to a model in which lipid molecules located on the transmembrane surface of a membrane protein (the annular lipid molecules), by playing a dominant role in the interaction between a membrane protein and the surrounding lipid bilayer, could effectively buffer the membrane protein from changes in the properties of the bulk lipid bilayer.     

Protein shape change has a major effect on the gating energy of a mechanosensitive channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k_BT to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k_BT on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.     

The estimation of distances between specific backbone-labeled sites in DNA using fluorescence resonance energy transfer.

A series DNA helices of twenty-four base pairs has been prepared for the study of fluorescence resonance energy transfer. Each of the DNA helices contains two phosphorothioate diesters (one in each strand) at pre-selected sites for introduction of the desired donor and acceptor fluorophores. The phosphorothioate-containing oligodeoxynucleotides have been prepared as pure Rp or Sp derivatives or as deastereomeric mixtures. Fluorescein and eosin are employed as the respective donor and acceptor fluorophores. A series of donor-acceptor pairs was generated by labeling of the appropriate phosphorothioate diester with the desired fluorophore and annealing the two complementary DNA strands (one containing the acceptor and one containing the donor fluorophore) to form the double-stranded helix. The 24-mer helices containing two covalently attached fluorophores exhibited some thermal destabilization and the extent of this destabilization was dependent upon the stereochemical orientation of the fluorophore. The Sp derivatives direct the fluorophore out, away from the the DNA helix, while the Rp derivatives direct the fluorophore toward the major groove. As expected, the Sp labeled duplexes were more stable than the corresponding Rp labeled sequences. However, all of the duplex structures formed were stable under the conditions used to measure energy transfer. Energy transfer could be observed with these complexes from the quenching of the donor fluorescence in the presence of the acceptor fluorophore. Using F?rster's theories, distances separating the fluorophores could be calculated that were generally in reasonable agreement with the distances expected in an idealized B-form DNA helix. However anomalous results were obtained for one donor/acceptor pair where the expected distance was less than 20 A. Fluorescence anisotropy values determined in solutions of varying viscosity were quite high suggesting that the fluorophores did not experience complete freedom of movement when attached to the DNA helix.     

Fluorescence imaging of two-photon linear dichroism: cholesterol depletion disrupts molecular orientation in cell membranes

The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer (energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane.     

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.     

Investigating lipid composition effects on the mechanosensitive channel of large conductance (MscL) using molecular dynamics simulations

Previous experimental work has shown that the functional properties of the mechanosensitive channel of large conductance (MscL) are affected by variations in lipid composition. Here, we utilize molecular dynamics simulations of Mycobacterium tuberculosis MscL to investigate such lipid composition effects on a molecular level. In particular, two sets of simulations were performed. In the first, trajectories using lipids with different headgroups (phosphatidylcholine and phosphatidylethanolamine) were compared. Protein-lipid interactions were clearly altered by the headgroup changes, leading to conformational differences in the C-terminal region of M. tuberculosis MscL. In the second set of simulations, lipid tails were gradually shortened, thinning the membrane over a molecular dynamics trajectory. These simulations showed evidence of hydrophobic matching between MscL and the lipid membrane, as previously proposed. For all simulations, protein-lipid interaction energies in the second transmembrane region were correlated to mutagenic data, emphasizing the importance of lipid interactions for proper MscL function.     

Application of radiationless energy transfer for distance measurements across membranes

For several 2- or 3-dimensional configurations of stationary donors and acceptors on or near a spherical membrane shell the transition probabilities for radiationless energy transfer are calculated, using Förster's approximation obtained for Coulombic dipole-dipole interaction of the transition moments. It turns out that the difference in the refractive indices for membrane (lipid) and bulk phase (water) has only a small influence on the transition probabilities. Furthermore, the curvature of biological cell surfaces can be neglected, but affects the energy transfer across small vesicles. The ratio thickness/radius of small vesicles can be determined by measuring fluorescence quenching of excited donors by acceptors on the other side of the membrane.     

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07–0.2 (on a scale of 0–1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.     

Spectral fluctuation of a single fluorophore conjugated to a protein molecule

We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous buffer to methanol in both single-molecule and bulk measurements were similar, indicating that, even at the single molecule level, the fluorescence spectrum is sensitive to microenvironmental changes of fluorophores. The time dependence of the fluorescence spectra of fluorophores attached to S1 molecules solely showed a fluctuation with time over a time scale of seconds. Because the fluorescence spectra of the same fluorophores directly conjugated to a glass surface remained constant, the spectral fluctuation observed for the fluorophores attached to S1 is most likely due to slow spontaneous conformational changes in the S1 molecule. Thus, single-molecule fluorescence spectroscopy appears to be a powerful tool to study the dynamic behavior of single biomolecules.     

Binding of U1A protein to the 3' untranslated region of its pre-mRNA.

We have studied the global structure of the U1A 3' untranslated region (UTR) element using fluorescence resonance energy transfer. Comparison of a single UTR-box with a series of oligoadenine bulges indicates that the UTR-box introduces a significant kink into the axis of the RNA, and quantification of the results suggests an included bend angle of approximately 100 degrees (i.e. 80 degrees from linear). The complete 3'-UTR element is also severely kinked by the two UTR-boxes. We can observe binding of U1A protein to the 3'-UTR element by a change in the fluorescence anisotropy of Cy3 attached to one of the helical ends. In parallel with the binding, we observe a marked increase in fluoresence resonance energy transfer efficiency between fluorophores attached at the two 5' termini, indicating a significant change in global conformation induced by the binding of the protein.     

Distortion of DNA junctions imposed by the binding of resolving enzymes: a fluorescence study.

Junction-resolving enzymes are nucleases that are specific for the structure of the four-way DNA junction. The binding of RuvC of Escherichia coli and Hjc of Sulfolobus solfataricus can be followed by an increase in the fluorescence anisotropy of Cy3 terminally attached to one of the helical arms of a four-way junction. By contrast, there was no change in fluorescein anisotropy with the binding of single dimers of these proteins. Fluorescence resonance energy transfer has therefore been used between fluorescein and Cy3 fluorophores attached to the ends of helical arms to analyse the global structure of the junction on protein binding. The results indicate that both enzymes induce a marked change in the global DNA conformation on the binding of a single dimer. The structure of the protein-junction complexes is independent of the presence or absence of divalent metal ions, unlike that of the protein-free junction. The structures of the RuvC and Hjc complexes are different, but both represent a significant opening of the structure compared to the stacked X-structure of the protein-free junction in the presence of magnesium ions. This protein-induced opening is likely to be important in the function of these enzymes.     

Membrane interactions of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKε) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids as a putative membrane anchor. To model the conformation, and stoichiometry of this segment in membrane-mimetic environments, we have prepared a peptide corresponding to this hydrophobic segment of DGKε of sequence KKKKLILWTLCSVLLPVFITFWKKKKK-NH₂. Flanking Lys residues mimic the natural setting of this peptide in DGKε, while facilitating peptide synthesis and characterization. Circular dichroism and fluorescence spectroscopic analysis demonstrated that the peptide has increased helical content and significant blue shifts in the presence of anionic - but not zwitterionic - bilayer membranes. When labeled with fluorophores that can undergo fluorescence resonance energy transfer, the peptide was found to dimerize - a result also observed from migration rates on SDS-PAGE gels under both reducing and non-reducing disulfide bridge conditions. The peptide was shown to preferentially interact with cholesterol in lipid films comprised of homogeneous mixtures of cholesterol and phosphatidylcholine, yet the presence of cholesterol in hydrated vesicle bilayers decreases its helical content. The peptide was also able to inhibit the activity of DGKε protein in vitro. Our overall findings suggest that the peptide ultimately cannot leave the bulk water for attachment/insertion into the outer leaflet of an erythrocyte-like bilayer, yet its core sequence is sufficiently hydrophobic to insert into membrane core regions when membrane attachment is promoted by electrostatic attraction to anionic lipid head groups of the inner leaflet of an erythrocyte-like bilayer.     

Association of daunomycin to membrane domains studied by fluorescence resonance energy transfer

1,6-Diphenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene are fluorophores used to explore different hydrophobic domains of membrane bilayers (Andrich, M.P. and Vanderkooi, J.M. (1976) Biochemistry 15, 1257-1265; Prendergast, F.G., Haugland, R.P. and Callahan, P.J. (1981) Biochemistry 20, 7333-7338). Fluorescence resonance energy transfer between these fluorophores, acting as energy donors, and the anthracycline, daunomycin, as the acceptor, was used to analyze the interaction of the drug with natural membranes, and its relative location within the membrane bilayer. The transfer process was demonstrated by: (1) emission fluorescence of the acceptor when the samples were excited at the excitation maximum of the donor (360 nm); and (2) progressive quenching of the energy donor (at 428 nm) when in the presence of increasing acceptor concentration. Also, the disruption of the energy transfer by solubilization of the membrane with Triton X-100 evidences a role for the membrane in providing the appropriate site(s) for energy transfer to occur. At moderately low daunomycin/membrane lipid ratios, the different efficiencies of resonance energy transfer between the two donors and daunomycin predicts a preferential, but not exclusive, location of the drug at membrane 'surface' domains, i.e., those regions of the bilayer explored by the 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probe. In support of this observation, a large fraction (approx. 75%) of membrane-associated daunomycin was rapidly sequestered away from the membrane upon addition of excess DNA, which forms high-affinity complexes with daunomycin (Chaires, J.B., Dattagupta, n. and Crothers, D.M. (1982) Biochemistry 21, 3927-3932), thus acting as a drug 'sink'. Also, a large fraction of drug was accessible to fluorescence quenching by iodide, a collisional water-soluble quencher. On the other hand, a smaller population of the membrane-associated daunomycin was characterized by slow sequestering by the added DNA and inaccessibility to quenching by iodide. We conclude that the daunomycin, which is only slowly sequestered, is located deep within the hydrophobic domains of the bilayer, likely to be those probed by 1,6-diphenyl-1,3,5-hexatriene.     

Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.     

Bilayer interactions of pHLIP, a peptide that can deliver drugs and target tumors

The pH-dependent insertion of pHLIP across membranes is proving to be a useful property for targeting acidic tissues or tumors and delivering drugs attached to its C-terminus. It also serves as a model peptide for studies of protein insertion into membranes, so further elucidation of the insertion mechanism of pHLIP and its features is desirable. We examine how the peptide perturbs a model phosphatidylcholine membrane and how it associates with the lipid bilayer using an array of fluorescence techniques, including fluorescence anisotropy measurements of TMA-DPH anchored in bilayers, quenching of pHLIP fluorescence by brominated lipids and acrylamide, and measurements of energy transfer between aromatic residues of pHLIP and TMA-DPH. When pHLIP is bound to the surface of bilayers near neutral pH, the membrane integrity is preserved whereas the elastic properties of bilayers are changed as reported by an increase of membrane viscosity. When it is inserted, there is little perturbation of the lipids. The results also suggest that pHLIP can bind to the membrane surface in a shallow or a deep mode depending on the phase state of the lipids. Using parallax analysis, the change of the penetration depth of pHLIP was estimated to be 0.4 Å from the bilayer center and 2.8 Å from the membrane surface after the liquid-to-gel phase transition.     

Sphingomyelin composition and physical asymmetries in native acetylcholine receptor-rich membranes

Selective enzymatic hydrolysis, lipid compositional analyses, and fluorescence studies have been carried out on acetylcholine receptor (AChR)-rich membranes from Torpedinidae to investigate the topology of sphingomyelin (SM) in the native membrane and its relationship with the AChR protein. Controlled sphingomyelinase hydrolysis of native membranes showed that SM is predominantly (approximately 60%) localized in the outer half of the lipid bilayer. Differences were also observed in the distribution of SM fatty acid molecular species in the two bilayer leaflets. A fluorescent SM derivative ( N-[10-(1-pyrenyl)decanoyl]sphingomyelin; Py-SM) was used to study protein-lipid interactions in the AChR-rich membrane and in affinity-purified Torpedo AChR reconstituted in liposomes made from Torpedo electrocyte lipid extracts. The efficiency of F?rster resonance energy transfer (FRET) from the protein to the pyrenyl-labeled lipid as a function of acceptor surface density was used to estimate distances and topography of the SM derivative relative to the protein. The dynamics of the lipid acyl chains were explored by measuring the thermal dependence of Py-SM excimer formation, sensitive to the fluidity of the membrane. Differences were observed in the concentration dependence of excimer/monomer pyrenyl fluorescence when measured by direct excitation of the probe as against under FRET conditions, indicating differences in the intermolecular collisional frequency of the fluorophores between bulk and protein-vicinal lipid environments, respectively. Py-SM exhibited a moderate selectivity for the protein-vicinal lipid domain, with a calculated relative affinity K(r) approximately 0.55. Upon sphingomyelinase digestion of the membrane, FRET efficiency increased by about 50%, indicating that the resulting pyrenyl-ceramide species have higher affinity for the protein than the parental SM derivative.