Lifetime fluorescence method for determining membrane topology of proteins

Recently, we introduced a sensitive method for determining the bilayer topology (cis- or trans-leaflet location) of single-site cysteine-linked 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorescent labels on membrane proteins. It uses a novel quencher, LysoUB, composed of a single acyl chain attached to a UniBlue chromophore. In its original version, the method relied on the comparison of steady-state fluorescence measurements of membrane-inserted proteins in samples with different distributions of the LysoUB in cis- and trans-leaflets of the lipid bilayer. Here we modify the method to take advantage of the fluorescence lifetime methodology, which allows us to simplify sample manipulation and, as a result, increase the reliability of topology determination. We tested the method using three model systems with artificially created all-cis, all-trans, and isotropic distribution of NBD. Because the quenching efficiency is higher when LysoUB and NBD are in the same leaflet, introduction of the quencher into the cis-leaflet results in a predictably different amount of quenching for these three model systems. Indeed, the addition of 2% LysoUB into the all-cis NBD model system causes strong reduction of the longest lifetime (from 8.1 to 4.9 ns), whereas the same addition of LysoUB results in marginal quenching (from 8.7 to 8.5 ns) in the case of all-trans NBD. This difference provides a good basis for topology determination using time-resolved fluorescence quenching.     

Membrane insertion and dissociation processes of a model transmembrane helix

An important subject for elucidating membrane protein (MP) folding is how transmembrane helices (TMHs) insert into and dissociate from membranes. We investigated helix dissociation kinetics and insertion topology by means of intervesicular transfer of the fluorophore-labeled completely hydrophobic model transmembrane helix NBD-(LALAAAA)(3)-NH(2) (NBD = 7-nitro-2-1,3-benzoxadiazol-4-yl). The peptide forms a topologically stable transmembrane helix, which is in a monomer-antiparallel dimer equilibrium [Yano, Y., Takemoto, T., Kobayashi, S., Yasui, H., Sakurai, H., Ohashi, W., Niwa, M., Futaki, S., Sugiura, Y., and Matsuzaki, K. (2002) Biochemistry 41, 3073-3080]. The helix transfer kinetics, representing the helix dissociation process, was monitored by fluorescence recovery of the quenched peptide in donor vesicles containing a quencher upon its transfer to acceptor vesicles without the quencher. The transfer kinetics and vesicle concentration dependence demonstrated that the transfer was mediated by monomer in the aqueous phase. Furthermore, the activation enthalpy was estimated to be +17.7 +/- 1.3 kcal mol(-1). Helix insertion topology, detected by chemical quenching of the NBD group in the outer leaflet by dithionite ions, was found to be controlled by transmembrane electric potential-helix macro dipole interaction. On the basis of these observations, a model for the helix insertion/dissociation processes was discussed.     

Monitoring the looping up of acyl chain labeled NBD lipids in membranes as a function of membrane phase state

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. The NBD group of acyl chain labeled NBD lipids is known to loop up to the membrane interface in fluid phase membranes. However, the organization of these lipids in gel phase membranes is not resolved. In this paper, we monitored the influence of the membrane phase state on the looping up behavior of acyl chain labeled NBD lipids utilizing red edge excitation shift (REES) and other sensitive fluorescence approaches. Interestingly, our REES results indicate that NBD group of lipids, which are labeled at the fatty acyl region, resides in the more hydrophobic region in gel phase membranes, and complete looping of the NBD group occurs only in the fluid phase. This is supported by other fluorescence parameters such as polarization and lifetime. Taken together, our results demonstrate that membrane packing, which depends on temperature and the phase state of the membrane, significantly affects the localization of acyl chain labeled NBD lipids. In view of the wide ranging use of NBD-labeled lipids in cell and membrane biology, these results could have potentially important implications in future studies involving these lipids as tracers.     

Membrane insertion pathway of annexin B12: thermodynamic and kinetic characterization by fluorescence correlation spectroscopy and fluorescence quenching

Experimental determination of the free energy stabilizing the structure of membrane proteins in their native lipid environment is undermined by the lack of appropriate methods and suitable model systems. Annexin B12 (ANX) is a soluble protein which reversibly inserts into lipid membranes under mildly acidic conditions, which makes it a good experimental model for thermodynamic studies of folding and stability of membrane proteins. Here we apply fluorescence correlation spectroscopy for quantitative analysis of ANX partitioning into large unilamellar vesicles containing either 25% or 75% anionic lipids. Membrane binding of ANX results in changes of autocorrelation time and amplitude, both of which are used in quantitative analysis. The thermodynamic scheme describing acid-induced membrane interactions of ANX considers two independent processes: pH-dependent formation of a membrane-competent form near the membrane interface and its insertion into the lipid bilayer. Our novel fluorescence lifetime topology method demonstrates that the insertion proceeds via an interfacial refolded intermediate state, which can be stabilized by anionic lipids. Lipid titration measurements are used to determine the free energy of both transmembrane insertion and interfacial penetration, which are found to be similar, approximately -10-12 kcal/mol. The formation of the membrane-competent form, examined in a lipid saturation experiment, was found to depend on the local proton concentration near the membrane interface, occurring with pK = 4.3 and involving the protonation of two residues. Our results demonstrate that fluorescence correlation spectroscopy is a convenient tool for the quantitative characterization of the energetics of transmembrane insertion and that pH-triggered ANX insertion is a useful model for studying the thermodynamic stability of membrane proteins.     

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts

Reliable protocols were established for investigating asymmetric distributions of 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-caproyl (C6NBD) phospholipids in the plasma membrane of boar sperm cells under physiological conditions. A method based on fluorescence resonance energy transfer was used to ensure that incorporation of the fluorescent phospholipids into the sperm proceeded via monomeric transfer. The total amount of incorporated phospholipid fluorescence and the proportion of translocated phospholipid fluorescence were determined by flow cytometric analysis before, and after, dithionite destruction of outer leaflet fluorescence. Catabolism of incorporated fluorescent phospholipids was blocked with phenylmethylsulfonyl fluoride. Membrane-damaged cells were detected with impermeant DNA stains, thereby enabling their exclusion from subsequent analyses of the flow cytometric data, whence it could be demonstrated that the labeled phospholipids were incorporated only via the outer plasma membrane leaflet in living sperm cells. Phospholipid uptake and internalization was followed at 38 degrees C. After 1 hr of labeling, about 96% of the incorporated C6NBD-phosphatidylserine, 80% of C6NBD-phosphatidylethanolamine, 18% of C6NBD-phosphatidylcholine, and 4% of C6NBD-sphingomyelin were found to have moved across the plasma membrane bilayer to the interior of the spermatozoa. These inward movements of fluorescent phospholipids were ATP-dependent and could be blocked with sulfhydryl reagents. Movements from the inner to the outer leaflet of the sperm plasma membrane were minimal for intact fluorescent phospholipids, but were rapid and ATP-independent for fluorescent lipid metabolites. The described method enables, for the first time, assessment of changes in lipid asymmetry under fertilizing conditions.     

The myristoylated amino terminus of Galpha(i)(1) plays a critical role in the structure and function of Galpha(i)(1) subunits in solution

To determine the role of the myristoylated amino terminus of Galpha in G protein activation, nine individual cysteine mutations along the myristoylated amino terminus of Galpha(i) were expressed in a functionally Cys-less background. Thiol reactive EPR and fluorescent probes were attached to each site as local reporters of mobility and conformational changes upon activation of Galpha(i)GDP by AlF(4)(-), as well as binding to Gbetagamma. EPR and steady state fluorescence anisotropy are consistent with a high degree of immobility for labeled residues in solution all along the amino terminus of myristoylated Galpha(i). This is in contrast to the high mobility of this region in nonmyristoylated Galpha(i) [Medkova, M., et al. (2002) Biochemistry 41, 9962-9972]. Steady state fluorescence measurements revealed pronounced increases in fluorescence upon activation for residues 14-17 and 21 located midway through the 30-amino acid stretch comprising the amino-terminal region. Collectively, the data suggest that myristoylation is an important structural determinant of the amino terminus of Galpha(i) proteins.     

Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles

A method for producing lipid vesicles containing fluorescent phospholipid analogues localized to the inner leaflet of their membrane was developed. Incubation of a 450-fold molar excess of serum albumin with lipid vesicles symmetrically labeled with 1 mol % 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazolyl)amino-caproyl phosphatidylcholine resulted in the removal of 99% of the fluorescent lipid from the outer leaflet. Asymmetrically labeled vesicles were separated from albumin/lipid complexes by gel filtration chromatography. Vesicles prepared in this manner were unable to transfer fluorescent lipid to cells during liposome-cell incubations. Liposomes asymmetrically labeled with other 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-phospholipid analogues were also prepared. Removal of amino-dodecanoyl-NBD-labeled lipids from the outer leaflet of liposomes required three times more bovine serum albumin, and 48 h of incubation. This method can be used to produce large amounts of asymmetrically labeled liposomes suitable for use in investigating a variety of membrane phenomena.     

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

Rapid topology probing using fluorescence spectroscopy in planar lipid bilayer: the pore-forming mechanism of the toxin Cry1Aa of Bacillus thuringiensis

Pore-forming toxins, many of which are pathogenic to humans, are highly dynamic proteins that adopt a different conformation in aqueous solution than in the lipid environment of the host membrane. Consequently, their crystal structures obtained in aqueous environment do not reflect the active conformation in the membrane, making it difficult to deduce the molecular determinants responsible for pore formation. To obtain structural information directly in the membrane, we introduce a fluorescence technique to probe the native topology of pore-forming toxins in planar lipid bilayers and follow their movement during pore formation. Using a Förster resonance energy transfer (FRET) approach between site-directedly labeled proteins and an absorbing compound (dipicrylamine) in the membrane, we simultaneously recorded the electrical current and fluorescence emission in horizontal planar lipid bilayers formed in plastic chips. With this system, we mapped the topology of the pore-forming domain of Cry1Aa, a biological pesticide from Bacillus thuringiensis, by determining the location of the loops between its seven α helices. We found that the majority of the toxins initially traverse from the cis to the trans leaflet of the membrane. Comparing the topologies of Cry1Aa in the active and inactive state in order to identify the pore-forming mechanism, we established that only the α3–α4 hairpin translocates through the membrane from the trans to the cis leaflet, whereas all other positions remained constant. As toxins are highly dynamic proteins, populations that differ in conformation might be present simultaneously. To test the presence of different populations, we designed double-FRET experiments, where a single donor interacts with two acceptors with very different kinetics (dipicrylamine and oxonol). Due to the nonlinear response of FRET and the dynamic change of the acceptor distribution, we can deduce the distribution of the acceptors in the membrane from the time course of the donor fluorescence. We found that Cry1Aa is present on both membrane leaflets.     

Organization and dynamics of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: a fluorescence approach

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. NBD-labeled lipids have previously been used to monitor the organization and dynamics of molecular assemblies such as membranes, micelles and reverse micelles utilizing the wavelength-selective fluorescence approach. In this paper, we have characterized the organization and dynamics of various NBD-labeled lipids using red edge excitation shift (REES) and other fluorescence approaches which include analysis of membrane penetration depths of the NBD group using the parallax method. We show here that the environment and location experienced by the NBD group of the NBD-labeled lipids could depend on the ionization state of the lipid. This could have potentially important implications in future studies involving NBD-labeled lipids as tracers in a cellular context.     

Identification of Protein-Protein and Protein-Ribosome Interacting Regions of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L

The mammalian mitochondrial inner membrane protein Oxa1L is involved in the insertion of a number of mitochondrial translation products into the inner membrane. During this process, the C-terminal tail of Oxa1L (Oxa1L-CTT) binds mitochondrial ribosomes and is believed to coordinate the synthesis and membrane insertion of the nascent chains into the membrane. The C-terminal tail of Oxa1L does not contain any Cys residues. Four variants of this protein with a specifically placed Cys residue at position 4, 39, 67, or 94 of Oxa1L-CTT have been prepared. These Cys residues have been derivatized with a fluorescent probe, tetramethylrhodamine-5-maleimide, for biophysical studies. Oxa1L-CTT forms oligomers cooperatively with a binding constant in the submicromolar range. Fluorescence anisotropy and fluorescence lifetime measurements indicate that contacts near a long helix close to position 39 of Oxa1L-CTT occur during oligomer formation. Fluorescence correlation spectroscopy measurements demonstrate that all of the Oxa1L-CTT derivatives bind to mammalian mitochondrial ribosomes. Steady-state fluorescence quenching and fluorescence lifetime data indicate that there are extensive contacts between Oxa1L-CTT and the ribosome-encompassing regions around positions 39, 67, and 94. The results of this study suggest that Oxa1L-CTT undergoes conformational changes and induced oligomer formation when it binds to the ribosome.     

Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter

The P-glycoprotein multidrug transporter acts as an ATP-powered efflux pump for a large variety of hydrophobic drugs, natural products, and peptides. The protein is proposed to interact with its substrates within the hydrophobic interior of the membrane. There is indirect evidence to suggest that P-glycoprotein can also transport, or "flip", short chain fluorescent lipids between leaflets of the membrane. In this study, we use a fluorescence quenching technique to directly show that P-glycoprotein reconstituted into proteoliposomes translocates a wide variety of NBD lipids from the outer to the inner leaflet of the bilayer. Flippase activity depended on ATP hydrolysis at the outer surface of the proteoliposome, and was inhibited by vanadate. P-Glycoprotein exhibited a broad specificity for phospholipids, and translocated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. Lipid derivatives that were flipped included molecules with long, short, unsaturated, and saturated acyl chains and species with the NBD group covalently linked to either acyl chains or the headgroup. The extent of lipid translocation from the outer to the inner leaflet in a 20 min period at 37 degrees C was directly estimated, and fell in the range of 0.36-1.83 nmol/mg of protein. Phospholipid flipping was inhibited in a concentration-dependent, saturable fashion by various substrates and modulators, including vinblastine, verapamil, and cyclosporin A, and the efficiency of inhibition correlated well with the affinity of binding to Pgp. Taken together, these results suggest that P-glycoprotein carries out both lipid translocation and drug transport by the same path. The transporter may be a generic flippase for hydrophobic molecules with the correct steric attributes that are present within the membrane interior.     

EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy

The green fluorescent protein (GFP) has become an invaluable marker for monitoring protein localization and gene expression in vivo. Recently a new red fluorescent protein (drFP583 or DsRed), isolated from tropical corals, has been described [Matz, M.V. et al. (1999) Nature Biotech. 17, 969-973]. With emission maxima at 509 and 583 nm respectively, EGFP and DsRed are suited for almost crossover free dual color labeling upon simultaneous excitation. We imaged mixed populations of Escherichia coli expressing either EGFP or DsRed by one-photon confocal and by two-photon microscopy. Both excitation modes proved to be suitable for imaging cells expressing either of the fluorescent proteins. DsRed had an extended maturation time and E. coli expressing this fluorescent protein were significantly smaller than those expressing EGFP. In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy.     

Analysis of membrane topology of neutral sphingomyelinase 2

Neutral sphingomyelinase 2 (nSMase2), which has two hydrophobic segments at its NH(2)-terminus, plays an important role in ceramide-mediated cell regulation. Here, we investigated the membrane topology of nSMase2. When a double-tagged nSMase2 at both the NH(2) and COOH termini, was overexpressed in MCF-7 cells, the signals from both tags were detected in the inner leaflet of the plasma membrane. Furthermore, insertion of a tag into the internal sequence and green fluorescent protein-fused deletion mutants revealed that the entire catalytic region of the protein was located on the cytosolic face of the membranes and each hydrophobic segment is integrated into the membranes, but unlikely to span the entire membrane. These results indicate the presence of the enzyme in the inner leaflet of plasma membrane.     

Conformational equilibrium of the reactive center loop of antithrombin examined by steady state and time-resolved fluorescence measurements: consequences for the mechanism of factor Xa inhibition by antithrombin-heparin complexes

Activation of antithrombin by high-affinity heparin as an inhibitor of factor Xa has been ascribed to an allosteric switch between two conformations of the reactive center loop. However, we have previously shown that other, weaker binding, charged polysaccharides can give intermediate degrees of activation [Gettins, P. G. W., et al. (1993) Biochemistry 32, 8385-8389]. To examine whether such intermediate activation results from different reactive center loop conformations or, more simply, from a different equilibrium constant between the same two extreme conformations, we have used NBD covalently bound at the P1 position of an engineered R393C variant of antithrombin as a fluorescent reporter group and measured fluorescence lifetimes of the label in free antithrombin as well as in antithrombin saturated with long-chain high-affinity heparin, high-affinity heparin pentasaccharide, long-chain low-affinity heparin, and dextran sulfate. Steady state emission spectra, anisotropies, and dynamic quenching measurements were also recorded. We found that the large steady state fluorescence enhancements produced by binding of activators resulted from relief of a static quench of fluorescence of NBD in approximately 50% of the labeled antithrombin molecules rather than from any large change in lifetimes, and that similar lifetimes were found for NBD in all activated antithrombin-oligosaccharide complexes. Similar anisotropies and positions of the NBD emission maxima were also found in the absence and presence of activators. In addition, NBD was accessible to quenching agents in both the absence and presence of activators, with an at most 2-fold increase in quenching constants between these two extremes. The simplest interpretation of the partial static quench in the absence of activators, the different degrees of enhancement by different antithrombin activators, and the similar fluorescence properties and quenching behavior of the different states is that there are two distinct types of conformational equilibrium involving three distinct states of antithrombin, which we designate A, A', and B. A and A' represent low-affinity or inactive states of approximately equal energy, both having the hinge residues inserted into beta-sheet A. A is fluorescent, while A' is statically quenched. State B represents the activated loop-expelled conformation in which none of the NBD fluorophores are statically quenched, as a result of the loop, including the P1-NBD, moving away from the body of the antithrombin. Different activators are able to shift the equilibrium to the high-activity (B) state to different extents and hence give different degrees of measured activity, and different degrees of relief of static quench. The similar properties and accessibility of the NBD in the A and B conformations also indicate that the P1 side chain is not buried in the low-activity A conformation, suggesting that an earlier proposal that activation involves exposure of the P1 side chain cannot be the explanation for activation. As an alternative explanation, heparin activation may give access to an exosite on antithrombin for binding to factor Xa and hence be the principal basis for enhancement of the rate of inhibition.     

Reversible transition between the surface trimer and membrane-inserted monomer of annexin 12

Under mildly acidic conditions, annexin 12 (ANX) inserts into lipid membranes to form a transbilayer pore [Langen, R., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 14060]. In this study, we have addressed the question of the oligomeric state of ANX in this transbilayer conformation by means of Forster-type resonance energy transfer (FRET). Two single-cysteine mutants (K132C and N244C) were labeled with either Alexa-532 (donor) or Alexa-647 (acceptor). The labels were positioned at the sites thought to be on the cis side of the known transmembrane regions [Ladokhin, A. S., et al. (2002) Biochemistry 41, 13617]. If the pore were comprised of an annexin oligomer, efficient energy transfer should be observed. Fluorescence excitation spectra of several mixtures of donor- and acceptor-labeled ANX were recorded under various conditions. Spectroscopic hallmarks of oligomerization-related FRET were established by following a well-documented transition of ANX from the soluble monomer to surface trimer upon addition of calcium at neutral pH. These hallmarks, however, were not detected for the membrane-inserted form of ANX at pH 4.5, suggesting that the transbilayer form is a monomer. This implies that the pore is formed by several transmembrane regions of the same ANX molecule. FRET and other fluorescence experiments demonstrate that the transitions between the surface trimer and membrane-inserted monomer are reversible. This reversibility, in combination with the absence of oligomerization in the water-soluble and inserted state, makes ANX a good experimental model for thermodynamic studies of folding and stability of membrane proteins.     

Orientation and dynamics of melittin in membranes of varying composition utilizing NBD fluorescence

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. The organization of membrane-bound melittin has earlier been shown to be dependent on the physical state and composition of membranes. In this study, we covalently labeled the N-terminal (Gly-1) and Lys-7 of melittin with an environment-sensitive fluorescent probe, the NBD group, to monitor the influence of negatively charged lipids and cholesterol on the organization and dynamics of membrane-bound melittin. Our results show that the NBD group of melittin labeled at its N-terminal end does not exhibit red edge excitation shift in DOPC and DOPC/DOPG membranes, whereas the NBD group of melittin labeled at Lys-7 exhibits REES of approximately 8 nm. This could be attributed to difference in membrane microenvironment experienced by the NBD groups in these analogs. Interestingly, the membrane environment of the NBD groups is sensitive to the presence of cholesterol, which is supported by time-resolved fluorescence measurements. Importantly, the orientation of melittin is found to be parallel to the membrane surface as determined by membrane penetration depth analysis using the parallax method in all cases. Our results constitute the first report to our knowledge describing the orientation of melittin in cholesterol-containing membranes. These results assume significance in the overall context of the role of membrane lipids in the orientation and function of membrane proteins and peptides.     

Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet

The P-glycoprotein multidrug transporter carries out ATP-driven cellular efflux of a wide variety of hydrophobic drugs, natural products, and peptides. Multiple binding sites for substrates appear to exist, most likely within the hydrophobic membrane spanning regions of the protein. Since ATP hydrolysis is coupled to drug transport, the spatial relationship of the drug binding sites relative to the ATPase catalytic sites is of considerable interest. We have used a fluorescence resonance energy transfer (FRET) approach to estimate the distance between a bound substrate and the catalytic sites in purified P-glycoprotein. The fluorescent dye Hoechst 33342 (H33342), a high-affinity P-glycoprotein substrate, bound to the transporter and acted as a FRET donor. H33342 showed greatly enhanced fluorescence emission when bound to P-glycoprotein, together with a substantial blue shift, indicating that the drug binding site is located in a nonpolar environment. Cys428 and Cys1071 within the catalytic sites of P-glycoprotein were covalently labeled with the acceptor fluorophore NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole). H33342 fluorescence was highly quenched when bound to NBD-labeled P-glycoprotein relative to unlabeled protein, indicating that FRET takes place from the bound dye to NBD. The distance separating the bound dye from the NBD acceptor was estimated to be approximately 38 A. Transition-state P-glycoprotein with the complex ADP*orthovanadate*Co2+ stably trapped at one catalytic site bound H33342 with similar affinity, and FRET measurements led to a similar separation distance estimate of 34 A. Since previous FRET studies indicated that a fluorophore bound within the catalytic site was positioned 31-35 A from the interfacial region of the bilayer, the H33342 binding site is likely located 10-14 A below the membrane surface, within the cytoplasmic leaflet of the membrane, in both resting-state and transition-state P-glycoprotein.     

Dynamics of membrane penetration of the fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group attached to an acyl chain of phosphatidylcholine

Location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to a short (C6) or a long (C12) sn2 acyl chain of a phosphatidylcholine molecule was investigated by fluorescence and solid-state NMR spectroscopy. 2H NMR lipid chain order parameters indicate a perturbation of the phospholipid packing density in the presence of NBD. Specifically, a decrease of molecular order was found for acyl chain segments of the lower, more hydrophobic region. Molecular collision probabilities determined by 1H magic angle spinning nuclear Overhauser enhancement spectroscopy indicate a highly dynamic reorientation of the probe in the membrane due to thermal fluctuations. A broad distribution of the fluorophore in the lipid bilayer is observed with a preferential location in the upper acyl chain/glycerol region. The distribution of the NBD group in the membrane is quite similar for both the long- and the short-chain analog. However, a slight preference of the NBD group for the lipid-water interface is found for C12-NBD-PC in comparison with C6-NBD-PC. Indeed, as shown by dithionite fluorescence assay, the long-chain analog reacts more favorably with dithionite, indicating a better accessibility of the probe by dithionite present in the aqueous phase. Forces determining the location of the fluorophore in the lipid water interface are discussed.     

Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer

The mechanisms underlying the ability of the Rho-GDP dissociation inhibitor (RhoGDI) to elicit the release of Rho-related GTP-binding proteins from membranes is currently unknown. In this report, we have set out to address this issue by using fluorescence resonance energy transfer approaches to examine the functional interactions of the RhoGDI with membrane-associated Cdc42. Two fluorescence assays were developed to monitor the interactions between these proteins in real time. The first involved measurements of resonance energy transfer between N-methylanthraniloyl GDP (MantGDP) bound to Cdc42 and fluorescein maleimide covalently attached to cysteine 79 of RhoGDI (RhoGDI-FM). This assay allowed us to directly monitor the binding of RhoGDI to membrane-associated Cdc42. The second fluorescence assay involved measurements of resonance energy transfer between membrane-associated Cdc42-MantGDP and hexadecyl(amino) fluorescein that was randomly inserted into the membrane bilayer. This assay enabled us to directly monitor the (GDI-induced) release of Cdc42 from membranes. Analyses of the rates of change in the fluorescence of Cdc42-MantGDP, which serves as a resonance energy transfer donor in both of these assays, as a function of RhoGDI concentration suggests a two-step mechanism to explain the ability of RhoGDI to stimulate the release of Cdc42 from membranes. Specifically, we propose that the GDI first binds rapidly to membrane-associated Cdc42 and then a slower isomerization occurs which represents the rate-limiting step for the dissociation of the Cdc42-RhoGDI complex from membranes. We propose that this slow step in the observed kinetics reflects the time-course of translocation of the geranyl-geranyl lipid tail of Cdc42 from the outer leaflet of the membrane to the isoprenyl binding site observed in the previously reported NMR structure of the Cdc42-RhoGDI complex [Gosser et al. (1997) Nature 387, 814].