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* Centro de Química-Física Molecular, Instituto Superior Técnico, Lisbon, Portugal; Departamento de Química, Universidade de Évora, Évora, Portugal; and Laboratory of Biophysics, Wageningen University, Wageningen, The Netherlands
Correspondence: Address reprint requests to Luís M.S. Loura, Centro de Química-Física Molecular, Complexo I, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal. Tel.: 35-121-841-9219; Fax: 35-121-846-4455; E-mail: pclloura{at}alfa.ist.utl.pt.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Deviations from perfect hydrophobic matching at the protein-lipid interface create a tension arising from the exposure of hydrophobic residues or acyl-chains to the hydrophilic medium. It is considered that the protein-lipid system adapts to hydrophobic mismatch conditions through several alternative and nonexclusive strategies such as ordering or disordering of perturbed phospholipids (change in bilayer thickness), lipid phase transition to nonlamellar phases, protein oligomerization/aggregation (minimization of interface area), helices tilting or side-chain rotation of a helical terminal residue (reduction in effective hydrophobic length), change in protein orientation, and decrease in the bilayer partitioning of the protein (for reviews see Killian, 1998; Dumas et al., 1999).
In the case of proteins incorporated in lipid systems containing lipids with different electrostatic properties or hydrophobic lengths, selectivity to one lipid component at the protein-lipid interface or preferential phase partitioning (depending on the lipid miscibility) may occur (Dumas et al., 1997, Lehtonen and Kinnunen, 1997; Fahsel et al., 2002).
In this study, we focused on the process of lipid selectivity at the protein-lipid interface. The problem of protein-lipid selectivity quantification has been addressed mainly from electron spin resonance (ESR) studies (see Marsh and Horváth, 1998, for a review) or other techniques which focus only on the protein-lipid interface, like tryptophan fluorescence quenching by brominated phospholipids (Everett et al., 1986; Williamson et al., 2002; O'Keeffe et al., 2000). The results obtained from ESR studies agree well with an annular model for protein-lipid selectivity, in which only the first shell of lipids around the integral protein, and in direct contact with it, is significantly disturbed by the protein incorporation in the bilayer (Lee, 2003; Marsh and Horváth, 1998).
ESR results report the fraction of motionally restricted lipids, whereas fluorescence collisional quenching depends on molecular contact. On the other hand, fluorescence resonance energy transfer (FRET) only depends on donor-acceptor distances and is an alternative technique to quantify lipid selectivity. Gutierrez-Merino derived approximate analytical expressions for the average rate of FRET (
kT
) in membranes undergoing phase separation or protein aggregation (Gutierrez-Merino, 1981a,b) and extended this formalism to the study of protein-lipid selectivity (Gutierrez-Merino et al., 1987). His model has proved to be useful to the study of the lipid annulus around the oligomeric acetylcholine receptor (Bonini et al., 2002; Antollini et al., 1996). However, there are some limitations to the model, namely, the simplification that underlies the formalism, which consists of considering resonance energy transfer (RET) only to neighboring acceptor molecules. On the other hand, with the experimental observable being the average RET efficiency given by
 | (1) |
kT is not straightforward. It is proposed that if the setting of experimental conditions is such that E is low (namely, kT is much smaller than kD), then E 
kT/kD (Gutierrez-Merino, 1981a). However, accurate low RET efficiencies are difficult to measure experimentally.
In the present work a new FRET formalism for an annular model of protein-lipid selectivity is proposed, and used in the quantification of M13 major coat protein selectivity toward different phospholipids. M13 major coat protein is the main protein component of the filamentous bacteriophage M13 with 
2800 copies. It contains a single hydrophobic transmembrane segment of 20 amino-acid residues, apart from an amphipathic N-terminal arm and a heavily basic C-terminus with a high density of lysines (for reviews see Stopar et al., 2003; Hemminga et al., 1993).
The present study is separated in two sections. The first section focuses on the effect of hydrophobic length, and the selectivity of M13 toward 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) ((18:1)2-PE-NBD) was determined in unsaturated phosphatidylcholine bilayers of different acyl chain lengths (14:1, 18:1, and 22:1). Whereas for 18:1 chains the chain length matches the hydrophobic length of the protein, there is significant hydrophobic mismatch for the other lipids used. The second part deals with specificity of M13 major coat protein to different phospholipid headgroups, some zwitterionic and other negatively charged. The results are compared to the results from the other methodologies for quantification of protein-lipid selectivity. Conclusions on the validity of the annular model for the M13 coat protein interaction with lipids are obtained.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Coat protein isolation and labeling
The T36C mutant of the M13 major coat protein was grown, purified from the phage and labeled with DCIA as described previously (Spruijt et al., 1996). For the removal of free label, DNA and other coat proteins, the mixture was applied to a Superdex 75 prep-grade HR 16/50 column (Pharmacia, Amersham Biosciences, Piscataway, NJ) and eluted with 50 mM sodium cholate, 150 mM NaCl, and 10 mM Tris-HCl pH 8. Fractions with an A280/A260 absorption ratio >1.5 were collected and concentrated by Amicon filtration (Amicon, Millipore, Bedford, MA).
Coat protein reconstitution in lipid vesicles
The labeled protein mutant was reconstituted in DOPC ((18:1)2-PC), DMoPC ((14:1)2-PC), and DEuPC ((22:1)2-PC) vesicles using the cholate-dialysis method (Spruijt et al., 1989). The phospholipid vesicles were produced as follows: the chloroform from solutions containing the desired NBD labeled and unlabeled phospholipid amount was evaporated under a stream of dry N2 and last traces were removed by further evaporation under vacuum. The lipids were then solubilized in 50 mM sodium cholate buffer (150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA) at pH 8 by brief sonication (Branson 250 cell disruptor, Branson Ultrasonics, Danbury, CT) until a clear opalescent solution was obtained, and then mixed with the wild-type and labeled protein. Samples had a phospholipid concentration between 0.5 and 1 mM (phospholipid concentration was determined through the analysis of inorganic phosphate according to McClare, 1971) and the lipid to protein ratio (L/P) was always kept at 700. Dialysis was carried out at room temperature and in the dark, with a 100-fold excess buffer containing 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA at pH 8. The buffer was replaced five times every 12 h.
Fluorescence spectroscopy
Absorption spectroscopy was carried out with a Jasco V-560 spectrophotometer (Tokyo, Japan). The absorption of the samples was kept <0.1 at the wavelength used for excitation.
Steady-state fluorescence measurements were obtained with an SLM-Aminco 8100 Series 2 spectrofluorimeter (Rochester, NY; with double excitation and emission monochromators, MC400) in a right-angle geometry. The light source was a 450-W Xe arc lamp and for reference a Rhodamine B quantum counter solution was used. 5x5 mm quartz cuvettes were used. All measurements were performed at room temperature.
The quantum yield of DCIA-labeled protein was determined using quinine bisulfate dissolved in 1 N H2SO4 (
= 0.55; Eaton, 1988) as a reference.
Fluorescence decay measurements of DCIA were carried out with a time-correlated single-photon timing system, which is described elsewhere (Loura et al., 2000). Measurements were performed at room temperature. Excitation and emission wavelengths were 340 and 450 nm, respectively. The timescales used were between 3 and 12 ps/ch, depending on the amount of NBD-labeled phospholipid present in the sample. Data analysis was carried out using a nonlinear, least-squares iterative convolution method based on the Marquardt algorithm (Marquardt, 1963). The goodness of the fit was judged from the experimental 
2 value, weighted residuals, and autocorrelation plot.
In all cases, the probe florescence decay was complex and described by a sum of exponentials,
 | (2) |
i are the fluorescence lifetimes. |
THEORETICAL BACKGROUND |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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 | (3) |
is the donor lifetime-weighted quantum yield in the presence of acceptor and 
is the donor lifetime-weighted quantum yield in the absence of acceptor. In turn, lifetime-weighted quantum yields are defined by Lakowicz (1999) as
 | (4) |
 | (5) |
2 is the orientation factor, n is the refractive index of the medium, and D is the donor quantum yield. J is calculated as
 | (6) |
) is the normalized emission spectrum of the donor and 
() is the absorption spectrum of the acceptor. If the -units in Eq. 6 are nm, the calculated R0 in Eq. 5 has Å units (Berberan-Santos and Prieto, 1987).
Annular model for M13 coat protein selectivity toward phospholipids
To analyze the FRET results, a model for transmembrane protein selectivity toward phospholipids was derived. The model assumes two populations of energy transfer acceptors, one located in the annular shell around the protein and the other outside it. The donor fluorescence decay curve will have energy transfer contributions from both populations,
 | (7) |
annular and random are the FRET contributions arising from energy transfer to annular labeled lipids and to randomly distributed labeled lipids outside the annular shell, respectively.
The acceptors in the annular shell (Fig. 1) are at a constant distance (d) to the coumarin fluorophore located in the center of the transmembrane domain, and therefore we can assume that the energy transfer to each of these acceptors is described by the rate constant
 | (8) |
D is the donor lifetime (in the absence of acceptor). The NBD fluorophores in the acceptor probes used in this study (phospholipids labeled with NBD in the headgroup or in the acyl-chain) are assumed to be located in the bilayer surface. For the chain-labeled lipids, this is justified because the NBD group "loops up" to the surface when attached to the end of the phospholipids acyl-chain (Chattopadhyay, 1990). The donor fluorophore is labeled in the M13 major coat protein 36th residue, located near the center of the bilayer (Spruijt et al., 1996). Therefore, to calculate d it is necessary to estimate the average distance (l) between the donor plane (center of bilayer) and the acceptors planes (both leaflets), as well as the lateral separation between both probes inside the transmembrane protein-annular shell lipids complex. For DOPC bilayers the position of the NBD fluorophore (l) in the derivatized phospholipids has been calculated through the parallax method (Abrams and London, 1993), and it was 18.9 and 19.8 Å from the bilayer center for the phospholipids labeled at the headgroup and at the acyl-chain, respectively. These values agree with other studies which employed different techniques to obtain the fluorophore position (Wolf et al., 1992; Màzeres et al., 1996). The reason for a position of NBD closer to the surface of the membrane while labeled at the acyl-chain is probably the increase in flexibility that the C12 chain allows. The lateral separation between the probes was assumed to be 8 Å. The estimated d was then 20.5 and 21.4 Å for NBD derivatized in the headgroup and acyl-chain, respectively. For the DMoPC and DEuPC bilayers the values used for l were 15.4 and 22.4 Å, respectively, as for each additional carbon in the phospholipid chain in the liquid crystalline phase the bilayer thickness increases 1.75 Å (Lewis and Engelman, 1983).
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 | (9) |
 | (10) |
as
 | (11) |
n2 is the acceptor density in each leaflet, l is the distance between the plane of the donors and the planes of acceptors, and Re is the distance between the protein axis and the second lipid shell (exclusion distance for bulk-located acceptors). In the present system, l is the unlabeled lipid bilayer thickness, and the exclusion distance is 16 Å assuming a radii of 5 Å and 4.5 Å for the protein and the phospholipid, respectively; see Fig. 1 b). The value n2 must be corrected for the presence of labeled lipid in the annular region, which therefore is not part of the randomly distributed acceptors pool. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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= 0.41). Using Eqs. 5 and 6, and assuming 2 = 2/3 (the isotropic dynamic limit) and n = 1.4 (Davenport et al., 1985), R0 = 39.3 Å is obtained for the DCIA-NBD FRET pair (Fig. 2). The value 2 = 2/3 was used, because for fluorophores in the center of a liquid crystalline bilayer, the rotational freedom should be sufficiently high to randomize orientations (for a detailed discussion see Loura et al., 1996).
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The donor fluorescence intensities ratio (
), which is related to the energy transfer efficiency, decreases upon increasing the acceptor (Eq. 3). The results are presented in Fig. 3. The results of fitting the derived formalism to the data are also shown in this figure, and the corresponding KS values are summarized in Table 1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Due to their ß-sheet conformation, they are detected by CD spectroscopy, and because of the hydrophobic mismatch between the protein and DEuPC (longer) or DMoPC (shorter) lipids, it was possible that while incorporated in pure bilayers of these components, the M13 coat protein could be submitted to irreversible aggregation. This hypothesis has been ruled out for both lipids in recent studies (Meijer et al., 2001; Fernandes et al., 2003). Nevertheless, M13 major coat protein was shown recently by us to aggregate reversibly while in these conditions from a study of BODIPY labeled protein steady-state fluorescence emission (Fernandes et al., 2003). However, in that study much higher concentrations of protein were used when compared with the present one, and assuming the aggregation constants obtained in that work, only up to 5% at the very most of protein could be aggregated at the protein concentration used throughout this study. Therefore we can consider that our results report the phospholipid selectivity properties of the monomeric M13 major coat protein.
The fitting of the annular model for protein-lipid interactions to the FRET data (Figs. 3 and 4), converged always to KS values above 1 (Table 1), as the energy transfer efficiencies (
) are above the expected value for random distribution of the labeled phospholipids. As our annular model assumes a random distribution outside the protein-lipid interface (which should be true for one-component bilayers in the liquid crystalline phase; Loura et al., 1996), this result is rationalized as an increase in local concentration of probe in the lipid annular shell around the protein. For (18:1)2-PE-NBD probe in DOPC ((18:1)2-PC) bilayers (Fig. 3 A), the value of KS was 1.4, pointing to almost complete randomization of the probe distribution in the bilayer, and therefore identical selectivity to the DOPC lipid. This was expected, because the probe acyl-chains are identical to the unlabeled lipid and allow a perfect hydrophobic matching of the protein.
The results from Fig. 3, A–C, all report energy transfer efficiencies to the (18:1)2-PE-NBD probe, but in different bilayers of one lipid component. In DOPC bilayers the value of KS was 1.4 as discussed above, but in DMoPC ((14:1)2-PC) and DEuPC ((22:1)2-PC) bilayers the relative association constant values were 2.9 and 2.1, respectively, confirming a greater selectivity toward the hydrophobic matching unlabeled phospholipid (DOPC).
M13 coat protein selectivity toward phospholipids with different headgroups
Results from analysis of the data on M13 coat protein selectivity toward phospholipids headgroups are presented in Fig. 4. Clearly the anionic-labeled phospholipids exhibit larger selectivity to the lipid annular region around the protein, especially the 18:1-(12:0-NBD)-PA and 18:1-(12:0-NBD)-PS probes (KS = 3.0 and 2.7, respectively). The 18:1-(12:0-NBD)-PG probe presents an intermediate selectivity (KS = 2.3), whereas 18:1-(12:0-NBD)-PC and 18:1-(12:0-NBD)-PE have identical relative association constants (KS = 2.0). The selectivity for anionic phospholipids must be a consequence of electrostatic interaction of these with the highly basic C-terminal domain of the protein, which contains four lysines.
Overall the selectivity of annular lipid-M13 coat protein is not large, which is common for intrinsic membrane proteins (Lee, 2003). Additionally, it has been shown that selectivity of some proteins toward anionic lipids is significantly decreased in the presence of increasing ionic strength (Marsh and Horváth, 1998). In our case the ionic strength was kept high, because it is necessary to keep the protein in the monomeric state (Spruijt and Hemminga, 1991), and this further explains our results.
Phospholipid selectivity ESR studies have already been performed with aggregated forms of M13 major coat protein (Peelen et al., 1992; Wolfs et al., 1989, Datema et al., 1987), resulting in similar selectivity patterns of M13 coat protein to phospholipid headgroups. Peelen et al. (1992), using a identical buffer type, ionic strength, and pH to that used in the present study, obtained the following relative association constants ratios (KS(PX)/KS(PC)) of M13 coat protein incorporated in 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC): KS(PA)/KS(PC) = 1.6, KS(PS)/KS(PC) = 1.2, KS(PG)/KS(PC) = 1.1, and KS(PE)/KS(PC) = 1. Overall the selectivity pattern is the same, and the relative association constants ratios are almost identical. The M13 coat protein was aggregated in that study, and according to the authors the number of first shell sites was five, that is, for each protein only a maximum of five lipids could be motionally restricted due to contact with the protein surface. For a monomeric helix, however, a value of 12 should be expected (Marsh and Horváth, 1998), and that was the number used in our model for the data analysis. Therefore it is particularly interesting that the ratios of the relative association constants remain almost identical. Apparently protein aggregation lowers the selectivity degree of each protein for phospholipids only through a decrease of available area for protein-lipid contacts but the relative association ratio with phospholipids of different headgroups remains the same. Even though the protein presents higher selectivity for the NBD-labeled phospholipids than for DOPC (KS(PC) = 2.0) (possibly due to electrostatic interactions with the NBD at the bilayer interface), the result presented above clearly shows that the presence of NBD at the bilayer interface does not change significantly the relative association ratios of the phospholipids.
Sanders et al. (1992) were not able to determine the selectivity of the M13 coat protein monomeric species toward phospholipids using ESR, because the monomer was not able to produce a sufficiently long-living boundary shell of lipids that could be detected by ESR spectroscopy. The fact that it was possible to clearly quantify relative association constants using FRET in the present study presents this technique as an alternative to ESR in protein-lipid studies.
One important difference between the ESR and FRET techniques is that the latter is not restricted to the lipids adjacent to a given protein molecule. Not only labeled lipids in the first shell of lipids will be potential acceptors to a donor-labeled integral protein, but also the acceptors in the other lipid shells surrounding the protein will contribute to the final result. For that reason, this study also seems to confirm the hypothesis of selectivity to anionic phospholipids by the protein to largely confine itself to an annular shell of lipids in direct contact with the protein, in the case of the M13 major coat protein.
In case that the annular region would extend beyond this first shell, our FRET analysis methodology (based in transfer to a single annular shell and also to the bulk) would recover substantially larger values for the relative association constants. Moreover, as commented above, our recovered KS/KS(PC) match those obtained from ESR measurements, which only detects immobilization of annular lipids upon incorporation of protein. The existence of a single annular lipid layer for this protein might be related to the fact that it has a sole transmembrane segment.
The FRET methodology has three interesting features. First, by choosing donor-acceptor pairs with different Förster radii it is possible to specifically study mainly the first-shell of lipids or also the outside shells, as was the case in the present study. The joint analysis of results coming from these different donor-acceptor pairs could allow for an even more detailed description of the protein-lipid arrangement in more complex systems. In our study, the relatively large R0 value for the used donor-acceptor pair meant that the experimental quenching curves shown in both Figs. 3 and 4 look similar at first sight. Nevertheless it is impressive that the analysis methodology is able to retrieve significant Ks values. Of course, this methodology could still be improved by the use of a donor-acceptor pair with a smaller R0 value, closer to the distances under measurement. Second, the more economic character of fluorescence studies, which requires much smaller amounts of material than ESR, should be stressed. And third, although this model leads to a somewhat complex decay law (Eqs. 7, 10, and 11), it is actually not necessary to analyze the decay curves with this law to recover the relevant parameters, unlike in other FRET studies (e.g., of lipid phase separation; see Loura et al., 2001). The theoretical curves are conveniently simulated and integrated in a worksheet to calculate the theoretical FRET efficiencies. These can be matched to experimental values by varying the KS value (the sole unknown parameter). The experimental FRET efficiencies could also be obtained from steady-state data. In our case, we obtained them from integration of donor decay curves because these are less prone to artifacts (e.g., light scattering, inner filter effects, measurement of absolute intensities), which in any case, could in principle be corrected for in a steady-state experiment.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Submitted on January 21, 2004; accepted for publication March 5, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTHEORETICAL BACKGROUNDRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Antollini, S. S., M. A. Soto, I. B. de Romanelli, C. Gutiérrez-Merino, P. Sotomayor, and F. J. Barrantes. 1996. Biophys. J. 70:1275–1284.
Berberan-Santos, M. N., and M. J. E. Prieto. 1987. Energy transfer in spherical geometry. Application to micelles. J. Chem. Soc. Faraday Trans. 283:1391–1409.
Bonini, I. C., S. S. Antollini, C. Gutiérrez-Merino, and F. J. Barrantes. 2002. Sphingomyelin composition and physical asymmetries in native acetylcholine receptor-rich membranes. Eur. Biophys. J. 31:417–427.[CrossRef][Medline]
Chattopadhyay, A. 1990. Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes. Chem. Phys. Lipids. 53:1–15.[CrossRef][Medline]
Datema, K. P., C. J. A. M. Wolfs, D. Marsh, A. Watts, and M. A. Hemminga. 1987. Spin label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers. Biochemistry. 26:7571–7574.[CrossRef][Medline]
Davenport, L., R. E. Dale, R. H. Bisby, and R. B. Cundall. 1985. Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in model lipid bilayer membrane systems by resonance energy transfer. Biochemistry. 24:4097–4108.[CrossRef][Medline]
Dumas, F., M. C. Lebrun, and J.-F. Tocanne. 1999. Is the protein/lipid hydrophobic matching principle relevant to membrane organization and functions? FEBS Lett. 458:271–277.[CrossRef][Medline]
Dumas, F., M. M. Sperotto, M.-C. Lebrun, J.-F. Tocanne, and O. G. Mouritsen. 1997. Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers. Biophys. J. 73:1940–1953.
Eaton, D. F. 1988. Reference materials for fluorescence measurement. Pure Appl. Chem. 60:1107–1114.[CrossRef]
Everett, J., A. Zlotnick, J. Tennyson, and P. W. Holloway. 1986. Fluorescence quenching of cytochrome b5 in vesicles with an asymmetric transbilayer distribution of brominated phosphatidylcholine. J. Biol. Chem. 261:6725–6729.
Fahsel, S., E. M. Pospiech, M. Zein, T. L. Hazlet, E. Gratton, and R. Winter. 2002. Modulation of concentration fluctuations in phase-separated lipid membranes by polypeptide insertion. Biophys. J. 83:334–344.
Fernandes, F., L. M. S. Loura, M. Prieto, R. Koehorst, R. Spruijt, and M. A. Hemminga. 2003. Dependence of M13 major coat protein oligomerization and lateral segregation on bilayer composition. Biophys. J. 85:2430–2441.
Gutierrez-Merino, C. 1981a. Quantitation of the Förster energy transfer for two-dimensional systems. I. Lateral phase separation in unilamellar vesicles formed by binary phospholipids mixtures. Biophys. Chem. 14:247–257.[CrossRef][Medline]
Gutierrez-Merino, C. 1981b. Quantitation of the Förster energy transfer for two-dimensional systems. II. Protein distribution and aggregation state in biological membranes. Biophys. Chem. 14:259–266.[CrossRef][Medline]
Gutierrez-Merino, C., F. Munkonge, A. M. Mata, J. M. East, B. L. Levinson, R. M. Napier, and A. G. Lee. 1987. The position of the ATP binding site on the (Ca2+ + Mg2+)-ATPase. Biochim. Biophys. Acta. 897:207–216.[Medline]
Hemminga, M. A., J. C. Sanders, C. J. A. M. Wolfs, and R. B. Spruijt. 1993. Lipid-protein interactions involved in bacteriophage M13 infection. Protein Lipid Inter. New Compr. Biochem. 25:191–212.
Killian, J. A. 1998. Hydrophobic mismatch between proteins and lipids in membranes. Biochim. Biophys. Acta. 1376:401–416.[Medline]
Lakowicz, J. R. 1999. Principals of Fluorescence Spectroscopy, 2nd Ed. Kluwer Academic/Plenum Press, New York. Chap. 8.
Lee, A. G. 2003. Lipid-protein interactions in biological membranes: a structural perspective. Biochim. Biophys. Acta. 1612:1–40.[Medline]
Lehtonen, J. Y. A., and P. K. J. Kinnunen. 1997. Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease. Biophys. J. 72:1247–1257.
Lewis, B. A., and D. M. Engelman. 1983. Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles. J. Mol. Biol. 166:211–217.[Medline]
Loura, L. M. S., A. Fedorov, and M. Prieto. 1996. Resonance energy transfer in a model system of membranes: application to gel and liquid crystalline phases. Biophys. J. 71:1823–1836.
Loura, L. M. S., R. F. M. de Almeida, and M. Prieto. 2001. Detection and characterization of membrane microheterogeneity by resonance energy transfer. J. Fluorescence. 11:197–209.[CrossRef]
Loura, L. M. S., A. Fedorov, and M. Prieto. 2000. Membrane probe distribution heterogeneity: a resonance energy transfer study. J. Phys. Chem. B. 104:6920–6931.
Marquardt, D. W. 1963. An algorithm for least-squares estimation of non-linear parameters. J. Soc. Ind. Appl. Math. (SIAM J.). 11:431–441.[CrossRef]
Marsh, D., and L. I. Horváth. 1998. Structure, dynamics and composition of the lipid-protein interface. Perspectives from spin-labelling. Biochim. Biophys. Acta. 1376:267–296.[Medline]
Màzeres, S., V. Schram, J.-F. Tocanne, and A. Lopez. 1996. 7-Nitrobenz-2-oxa-1,3-diazole-4-yl-labeled phospholipids in lipid membranes: differences in fluorescence behavior. Biophys. J. 71:327–335.
McClare, C. 1971. An accurate and convenient organic phosphorus assay. Anal. Biochem. 39:527–530.[CrossRef][Medline]
Meijer, A. B., R. B. Spruijt, C. J. A. M. Wolfs, and M. A. Hemminga. 2001. Membrane-anchoring interactions of M13 major coat protein. Biochemistry. 40:8815–8820.[CrossRef][Medline]
Mouritsen, O. G., and M. Bloom. 1984. Mattress model of lipid-protein interactions in membranes. Biophys. J. 46:141–153.
O'Keeffe, A. H., J. M. East, and A. G. Lee. 2000. Selectivity in lipid binding to the bacterial outer membrane protein OmpF. Biophys. J. 79:2066–2074.
Peelen, S. J. C. J., J. C. Sanders, M. A. Hemminga, and D. Marsh. 1992. Biochemistry. Stoichiometry, selectivity and exchange dynamics of lipid-protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance. Effects of protein secondary structure. Biochemistry. 31:2670–2677.[CrossRef][Medline]
Sanders, J. C., M. F. Ottaviani, A. van Hoek, and A. J. W. G. Visser. 1992. A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers. Eur. Biophys. J. 21:305–311.[Medline]
Spruijt, R. B., and M. A. Hemminga. 1991. The in situ aggregational and conformational state of the major coat protein of bacteriophage M13 in phospholipid bilayers mimicking the inner membrane of host Escherichia coli. Biochemistry. 30:11147–11154.[CrossRef][Medline]
Spruijt, R. B., C. J. A. M. Wolfs, and M. A. Hemminga. 1989. Aggregation related conformational change of the membrane-associated coat protein of bacteriophage M13. Biochemistry. 28:9158–9165.[CrossRef][Medline]
Spruijt, R. B., C. J. A. M. Wolfs, J. W. G. Verver, and M. A. Hemminga. 1996. Accessibility and environment probing using cysteine residues introduced along the putative transmembrane domain of the major coat protein of bacteriophage M13. Biochemistry. 35:10383–10391.[CrossRef][Medline]
Stopar, D., R. B. Spruijt, C. J. A. M. Wolfs, and M. A. Hemminga. 2003. Biochim. Biophys. Acta. 1611:5–15.[Medline]
Williamson, I. M., S. J. Alvis, J. M. East, and A. G. Lee. 2002. Interactions of phospholipids with the potassium channel KcsA. Biophys. J. 83:2026–2038.
Wolf, D. E., A. P. Winiski, A. E. Ting, K. M. Bocian, and R. E. Pagano. 1992. Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer. Biochemistry. 31:2865–2873.[CrossRef][Medline]
Wolfs, C. L. A. M., L. I. Horváth, D. Marsh, A. Watts, and M. A. Hemminga. 1989. Spin label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers. Biochemistry. 28:995–1001.[CrossRef][Medline]
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