Membrane-anchoring interactions of M13 major coat protein.

The response to hydrophobic mismatch of membrane-bound M13 major coat protein is measured using site-directed fluorescence and ESR spectroscopy. For this purpose, we investigate the membrane-anchoring interactions of M13 coat protein in model systems consisting of phosphatidylcholine bilayers that vary in hydrophobic thickness. Mutant coat proteins are prepared with an AEDANS-labeled single cysteine residue in the hinge region of the protein or at the C-terminal side of the transmembrane helix. In addition, the fluorescence of the tryptophan residue is studied as a monitor for the N-terminal side of the transmembrane helix. The fluorescence results show that the hinge region and C-terminal side of the transmembrane helix hardly respond to hydrophobic mismatch. In contrast, the N-terminal side of the helical transmembrane domain shifts to a more apolar environment, when the hydrophobic thickness is increased. The apparent strong membrane-anchoring interactions of the C-terminus are confirmed using a mutant that contains a longer transmembrane domain. As a result of this mutation, the tryptophan residue at the N-terminal side of the helical domain clearly shifts to a more polar environment, whereas the labeled position 46 at the C-terminal side is not affected. The phenylalanines in the C-terminal part of the protein play an important role in these apparent strong anchoring interactions. This is demonstrated with a mutant in which both phenylalanines are replaced by alanine residues. The phenylalanine residues in the C-terminus affect the location in the membrane of the entire transmembrane domain of the protein.

Conformation and orientation of the gene 9 minor coat protein of bacteriophage M13 in phospholipid bilayers.

The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in model membrane systems, which varied in lipid head group and lipid acyl chain composition. By using FTIR spectroscopy and subsequent band analysis a quantitative analysis of the secondary structure of the protein was obtained. The secondary structure of the gene 9 protein predominantly consists of alpha-helical (67%) and turn (33%) structures. The turn structure is likely to be located C-terminally where it has a function in recognizing the phage DNA during bacteriophage assembly. Attenuated total reflection FTIR spectroscopy was used to determine the orientation of gene 9 protein in the membrane, revealing that the alpha-helical domain is mainly transmembrane. The conformational and orientational measurements result in two models for the gene 9 protein in the membrane: a single transmembrane helix model and a two-helix model consisting of a 15 amino acid long transmembrane helix and a 10 amino acid long helix oriented parallel to the membrane plane. Potential structural consequences for both models are discussed.

Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes.

Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane after phage infection of Escherichia coli. The way of insertion of this small protein (32 amino acids) into membranes is still unknown. Here we show that the protein is able to insert in monolayers. The limiting surface pressure of 35 mN/m for 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol lipid systems indicates that this spontaneous insertion can also occur in vivo. By carboxyfluorescein leakage experiments of vesicles it is demonstrated that protein monomers, or at least small aggregates, are more effective in releasing carboxyfluorescein than highly aggregated protein. The final orientation of the protein in the bilayer after insertion was addressed by proteinase K digestion, thereby making use of the unique C-terminal location of the antigenic binding site. After insertion the C-terminus is still available for the enzymatic digestion, while the N-terminus is not. This leads to the overall conclusion that the protein is able to insert spontaneously into membranes without the need of any machinery or transmembrane gradient, with the positively charged C-terminus remaining on the outside. The orientation after insertion of gene 9 protein is in agreement with the 'positive inside rule'.

Configurations of the N-terminal amphipathic domain of the membrane-bound M13 major coat protein.

The M13 major coat protein has been extensively studied in detergent-based and phospholipid model systems to elucidate its structure. This resulted in an L-shaped model structure of the protein in membranes. An amphipathic alpha-helical N-terminal arm, which is parallel to the surface of the membrane, is connected via a flexible linker to an alpha-helical transmembrane domain. In the present study, a fluorescence polarity probe or ESR spin probe is attached to the SH group of a series of N-terminal single cysteine mutants, which were reconstituted into DOPC model membranes. With ESR spectroscopy, we measured the local mobility of N-terminal positions of the protein in the membrane. This is supplemented with relative depth measurements at these positions by fluorescence spectroscopy via the wavelength of maximum emission and fluorescence quenching. Results show the existence of at least two possible configurations of the M13 amphipathic N-terminal arm on the ESR time scale. The arm is bound either to the membrane surface or in the water phase. The removal or addition of a hydrophobic membrane-anchor by site-specific mutagenesis changes the ratio between the membrane-bound and the water phase fraction.

Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13.

During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17-24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.

Membrane assembly of the bacteriophage Pf3 major coat protein.

The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.

Conformational and aggregational properties of the gene 9 minor coat protein of bacteriophage M13 in membrane-mimicking systems.

The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in various membrane-mimicking systems, including organic solvents, detergent micelles, and phospholipid bilayers. For this purpose we determined the conformational and aggregational properties of the chemically synthesized protein by CD, FTIR, and HPSEC. The protein appears to be in a monomeric or small oligomeric alpha-helical state in TFE but adopts a mixture of alpha-helical and random structure after subsequent incorporation into SDS or DOPG. When solubilized by sodium cholate, however, the protein undergoes a transition in time into large aggregates, which contain mainly beta-sheet conformation. The rate of this beta-polymerization process was decreased at lower temperature and higher concentrations of sodium cholate. This aggregation was reversed only upon addition of high concentrations of the strong detergent SDS. By reconstitution of the cholate-solubilized protein into DOPG, it was found that the state of the protein, whether initially alpha-helical monomeric/oligomeric or beta-sheet aggregate, did not change. On the basis of our results, we propose that the principal conformational state of membrane-bound gene 9 protein in vivo is alpha-helical.

Mimicking initial interactions of bacteriophage M13 coat protein disassembly in model membrane systems.

The structure and changes in environment of the M13 major coat protein were studied in model systems, mimicking the initial molecular process of the phage disassembly. For this purpose we have systematically studied protein associations with various detergents and lipids in two different coat protein assemblies: phage particles and S-forms. It is remarkable that the major coat protein can change its conformation to accommodate three distinctly different environments: phage filament, S-form, and membrane-bound form. The structural and environmental changes during this protein transformations were studied by site-directed spin labeling, fluorescence labeling, and CD spectroscopy in different membrane model systems. The phage particles were disrupted only by strong ionic detergents [sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide and (CTAB)] but were not affected by sodium cholate and sodium deoxycholate, nonionic detergents, and dilauroyl-l-alpha-phosphatidylcholine (DLPC) lipid bilayers. Conversion of the phage particles into S-forms by addition of chloroform rendered the coat protein accessible for the association with different ionic and nonionic detergents, as well as DLPC lipids. The disruption of the S-form by all detergents studied was instantaneous but was slower with DLPC vesicles. Only small unilamellar vesicles effectively solubilized the S-form. The data suggest that the viral protein coat is inherently unstable when the major coat protein is exposed to amphiphilic molecules. During conversion from the phage to the S-form, and subsequently to the membrane-bound form, the coat protein undergoes pronounced changes in environment, and in response the alpha-helix content decreases and the local protein structure changes dramatically. This adaptation of the protein conformation enables a stable association of the protein with the membrane.

In situ aggregational state of M13 bacteriophage major coat protein in sodium cholate and lipid bilayers.

The in situ aggregational behavior of the bacteriophage M13 major coat protein was determined for the protein isolated in sodium cholate and reconstituted into DOPC lipid bilayers. For this purpose, the cysteine mutants A49C and T36C of the major coat protein were labeled with either a maleimido spin-label or a fluorescence label (IAEDANS). The steric restrictions sensed by the spin-label were used to evaluate the local protein conformation and the extent of protein-protein interactions at the position of the labeled residue. In addition, fluorescent labels covalently attached to the protein were used to determine the polarity of the local environment. The labeled coat protein mutants were examined under different conditions of protein association (amphiphile environment, ionic strength, temperature, and pH). The aggregational state of the major coat protein solubilized from the phage particle in sodium cholate was not dependent on the ionic strength, but was strongly dependent on cholate concentration and pH during sample preparation. At pH 7.0 and high sodium cholate concentration, the protein was in a dimeric form. The unusually strong association properties of the protein dimer in sodium cholate at pH 7.0 were attributed to the inability of sodium cholate to disrupt the strong hydrophobic forces between neighboring protein subunits in the phage particle. Such a "structural protein dimer" was, however, completely and irreversibly disrupted at pH 10.0. Qualitatively the same aggregational tendency was found upon changing the pH for the coat protein reconstituted in DOPC lipid bilayers. This reveals that the dimer disruption process is primarily a protein property, because there are no titratable groups on DOPC in the experimental pH range. The results are interpreted in terms of a model relating the protein aggregational state in the assembled phage to the protein aggregational behavior in sodium cholate and lipid bilayers.

Conventional and saturation-transfer EPR of spin-labeled mutant bacteriophage M13 coat protein in phospholipid bilayers.

A mutant of bacteriophage M13 was prepared in which a cysteine residue was introduced at position 25 of the major coat protein. The mutant coat protein was spin-labeled with a nitroxide derivative of maleimide and incorporated at different lipid-to-protein (L/P) ratios in DOPC or DOPG. The rotational dynamics of the reconstituted mutant coat protein was studied using EPR and saturation transfer (ST) EPR techniques. The spectra are indicative for an anisotropic motion of the maleimide spin label with a high order parameter (S = 0.94). This is interpreted as a wobbling motion of the spin label with a correlation time of about 10(-6) to 10(-5) s within a cone, and a rotation of the spin label about its long molecular axis with a correlation time of about l0(-7) s. The wobbling motion is found to correspond generally to the overall rotational motion of a coat protein monomer about the normal to the bilayer. This motion is found to be sensitive to the temperature and L/P ratio. The high value of the order parameter implies that the spin label experiences a strong squeezing effect by its local environment, that reduces the amplitude of the wobbling motion. This squeezing effect is suggested to arise from a turn structure in the coat protein from Gly23 to Glu20.

Local dynamics of the M13 major coat protein in different membrane-mimicking systems.

The local environment of the transmembrane and C-terminal domain of M13 major coat protein was probed by site-directed ESR spin labeling when the protein was introduced into three membrane-mimicking systems, DOPC vesicles, sodium cholate micelles, and SDS micelles. For this purpose, we have inserted unique cysteine residues at specific positions in the transmembrane and C-terminal region, using site-directed mutagenesis. Seven viable mutants with reasonable yield were harvested: A25C, V31C, T36C, G38C, T46C, A49C, and S50C. The mutant coat proteins were indistinguishable from wild type M13 coat protein with respect to their conformational and aggregational properties. The ESR data suggest that the amino acid positions 25 and 46 of the coat protein in DOPC vesicles are located close to the membrane-water interface. In this way the lysines at positions 40, 43, and 44 and the phenylalanines at positions 42 and 45 act as hydrophilic and hydrophobic anchors, respectively. The ESR spectra of site specific maleimido spin-labeled mutant coat proteins reconstituted into DOPC vesicles and solubilized in sodium cholate or SDS indicate that the local dynamics of the major coat protein is significantly affected by its structural environment (micellar vs bilayer), location (aqueous vs hydrophobic), and lipid/protein ratio. The detergents SDS and sodium cholate sufficiently well solubilize the major coat protein and largely retain its secondary structure elements. However, the results indicate that they have a poorly defined protein-amphiphilic structure and lipid-water interface as compared to bilayers and thus are not a good substitute for lipid bilayers in biophysical studies.

Accessibility and environment probing using cysteine residues introduced along the putative transmembrane domain of the major coat protein of bacteriophage M13.

The major coat protein of the filamentous bacteriophage M13 is located in the inner membrane of host cell Escherichia coli prior to assembly into virions. To identify the transmembrane domain of the coat protein, we have introduced unique cysteine residues along the putative transmembrane domain at position 25, 31, 33, 36, 38, 46, 47, 49, or 50. The mutant major coat protein was solubilized by membrane-mimicking detergents or reconstituted into mixed bilayers of phospholipids. Information about the environmental polarity was deduced from the wavelength of maximum emission, using N-[[(iodoacetyl)-amino)ethyl]-1-sulfonaphthylamine (IAEDANS) attached to the SH groups of the cysteines as a fluorescent probe. Additional information was obtained by determining the accessibility of AEDANS for the fluorescence quencher molecules acrylamide and 5-doxylstearic acid, and the reactivity of the cysteine's sulfhydryl group toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Our data suggest transmembrane boundaries close to residue 25 and 46, with residue 25 inside the hydrophobic part of the membrane in very close proximity to the membrane-water interface and residue 46 located at the membrane-water interface. Domains of the mutant coat protein which are packed or coated by cholate molecules and various other detergents [except for sodium dodecyl sulfate (SDS)] are at least similarly packed by phospholipid molecules in bilayers. SDS is a good solubilizing detergent but badly mimics the typical nature of a membrane structure. The overall results are interpreted with respect to the established conformation of the coat protein and its membrane anchoring mechanism.

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.

The major coat protein of bacteriophage M13 has been reconstituted into phospholipids with a composition comparable to that found in the host (Escherichia coli) inner membrane. Reconstitution experiments have revealed conditions in which the alpha-oligomeric state is favored over the beta-polymeric state. Discrimination between the two states of the membrane-bound coat protein (alpha-oligomeric and beta-polymeric states) has been achieved using high-performance size-exclusion chromatography and circular dichroism. Interprotein electrostatic interactions, probably induced by head-tail binding, are initiated and facilitating the aggregation-related conformational change process, in which alpha-oligomeric coat protein is converted into beta-polymeric coat protein. A model for this beta-polymerization process of the coat protein is presented. The alpha-helical protein has been studied by the in situ Trp fluorescence quantum yield. This shows that the average distances between coat proteins decrease upon lowering the L/P ratio. In situ cross-linking reactions of the coat protein at high L/P ratios reveal a monomeric state, thus excluding specific aggregation of the coat protein. A monomeric state of detergent-solubilized coat protein is also observed using SDS-PAGE and SDS-HPSEC. On the basis of these results, the smallest in situ aggregational entity of the coat protein is proposed to be a monomer. This finding is discussed in relation to the functional state of the M13 coat protein in the membrane-bound assembly and disassembly processes during infection.

A NMR investigation on the interactions of the alpha-oligomeric form of the M13 coat protein with lipids, which mimic the Escherichia coli inner membrane.

The interaction of the M13 bacteriophage major coat protein in the alpha-oligomeric form with specifically deuterated phospholipid headgroups which mimic the Escherichia coli inner membrane, has been studied using NMR methods. As can be seen from the deuterium NMR spectra obtained with headgroup trimethyl deuterated DOPC, the coat protein in the alpha-oligomeric form does not give rise to trapped lipids as observed with M13 coat protein in the beta-polymeric form (Van Gorkom et al. (1990) Biochemistry 29, 3828-3834). The quadrupolar splittings of the alpha headgroup methylene deuterons of deuterated phosphatidylcholine and phosphatidylethanolamine decrease, whereas the quadrupolar splittings of the beta headgroup methylene deuterons of the two lipids increase with increasing protein content. All deuterated segments in the phosphatidylglycerol headgroup show the same relative decrease of the NMR quadrupolar splittings. These results are interpreted in terms of a change in torsion angles of the methylene groups, induced by positive charges, probably lysine residues of the protein at the membrane surface. For all lipid bilayer compositions studied the head-group perturbations are similar. It is concluded that there is no strong specific interaction between one of the lipid types examined and the M13 coat protein. From the spin-spin (T2e) relaxation time and spin-lattice (T1z) relaxation time of all deuterated lipids it is concluded that at the bilayer surface only slow motions are affected by the M13 coat protein.

Interaction of non-enveloped plant viruses and their viral coat proteins with phospholipid vesicles.

The interaction of the non-enveloped plant viruses TMV (rod-shaped) and CCMV (spherical) and of their coat proteins in several well-defined aggregation states, with artificial membranes was investigated to study the early stages of the cellular infection process. Information about the separate steps in the interaction mechanisms was obtained by employing three assays, performed as a function of vesicle size, net membrane charge, pH and ionic strength. The assays allow to discriminate between aggregation of vesicles (turbidity assay) and membrane destabilization (vesicle leakage assay and lipid mixing assay). The aggregation of the vesicles is a result of electrostatic interactions between the viral material and vesicles surface (cross-linking), while the destabilization of the membrane is a result of penetration or bilayer disruption by hydrophobic protein domains. TMV virions and its coat protein, and CCMV virions, due to their net negative charge, predominantly interact with positively charged membranes. The coat protein of CCMV was found to interact with negatively charged membranes, an interaction that can be assigned to its basical N-terminal sequence. Changing the aggregational state of the viral coat proteins yielded most significant interactions in case of TMV coat protein aggregated in the disk form and CCMV coat protein aggregated in empty capsids with oppositely charged membranes. These protein aggregates are found to be the best compromise between efficiency (capacity of the protein to bridge vesicles and destabilize their membranes) and concentration of protein aggregates. The results are discussed with respect to previously proposed biological models of the early stages of plant virus infection.

Aggregation-related conformational change of the membrane-associated coat protein of bacteriophage M13.

The state of the coat protein of bacteriophage M13, reconstituted into amphiphilic media, has been investigated. The in situ conformation of the coat protein has been determined by using circular dichroism. Minimum numbers for the protein aggregation in the system have been determined after disruption of the lipid-protein system and subsequent uptake of the protein in cholate micelles. The aggregational state and conformation of the protein were affected by (1) the method of coat protein isolation (phenol extraction vs cholate isolation), (2) the nature of amphiphiles used (variation in phospholipid headgroups and acyl chains), and (3) the ratio of amphiphiles and protein. Under all conditions, phenol-extracted coat protein was in a predominantly beta-structure and in a highly aggregated polymeric form. Cholate-isolated coat protein was initially oligomeric and contained a substantial amount of alpha-helix. Below an aggregation number of 20, this protein showed a reversible aggregation with no change in conformation. Upon further aggregation, a conformational change was observed, and aggregation was irreversible, resulting in predominantly beta-structured coat protein polymers. This effect was observed upon uptake in phospholipids at low lipid to protein molar ratios (L/P ratios) and with phosphatidylcholines (PC) and phosphatidic acids (PA) containing saturated acyl chains. After reconstitution in phospholipids with unsaturated acyl chains and with phosphatidylglycerols (PG) at high L/P ratios, the original alpha-helix-containing state of the coat protein was maintained. Cross-linking experiments demonstrated that the beta-polymers are able to form reversible superaggregates within the vesicle system. An aggregation-related conformational change mechanism for the coat protein in phospholipid systems is proposed.

Deuterium nuclear magnetic resonance investigation of bacteriophage M13 coat protein in dimyristoylphosphatidylcholine liposomes using palmitic acid as a probe.

The effect of incorporation of various amounts of M13 bacteriophage coat protein on the bilayer order and acyl chain motion in dimyristoylphosphatidylcholine (DMPC) liposomes has been investigated using deuterium NMR of specifically deuterated palmitic acid as a bilayer probe, phosphorus NMR and additional spin-label electron spin resonance (ESR). The secondary structure of the M13 coat protein in these bilayers was determined from circular dichroism spectra. Phosphorus NMR spectra of the mixed liposomes are characteristic for DMPC organized in bilayers, also after incorporation of various levels of M13 protein. Circular dichroism spectra of the coat protein indicate that the protein conformation is predominantly a beta-structure (more than 75%). Various incorporation levels of M13 coat protein do not affect the order of the deuterium-labelled positions along the acyl chain at the carbon-2, 9 and 16 positions. In contrast, the spin-spin relaxation times decrease at higher protein levels, especially at the carbon-16 position. The spin-label ESR spectra of the same system using 14-doxylstearic acid as a label show a second, motionally restricted component, that is not observed by deuterium NMR. The NMR and ESR results are consistent with a model in which the fatty acid molecules are in a fast two-site exchange (at a rate of approx. 10(7) Hz) between the sites in the bulk of the lipid bilayer and the motionally restricted sites on the coat protein.

Time-resolved tryptophan fluorescence anisotropy investigation of bacteriophage M13 coat protein in micelles and mixed bilayers.

Coat protein of bacteriophage M13 is examined in micelles and vesicles by time-resolved tryptophan fluorescence and anisotropy decay measurements and circular dichroism experiments. Circular dichroism indicates that the coat protein has alpha-helix (60%) and beta-structure (28%) in 700 mM sodium dodecyl sulfate micelles and predominantly beta-structure (94%) in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidic acid (80/20 w/w) small unilamellar vesicles. The fluorescence decay at 344 nm of the single tryptophan in the coat protein after excitation at 295 or 300 nm is a triple exponential. In the micelles the anisotropy decay is a double exponential. A short, temperature-independent correlation time of 0.5 +/- 0.2 ns reflects a rapid depolarization process within the coat protein. The overall rotation of the coat protein-detergent complex is observed in the decay as a longer correlation time of 9.8 +/- 0.5 ns (at 20 degrees C) and has a temperature dependence that satisfies the Stokes-Einstein relation. In vesicles at all lipid to protein molar ratios in the range from 20 to 410, the calculated order parameter is constant with a value of 0.7 +/- 0.1 from 10 to 40 degrees C, although the lipids undergo the gel to liquid-crystalline phase transition. The longer correlation time decreases gradually on increasing temperature. This effect probably arises from an increasing segmental mobility within the coat protein. The results are consistent with a model in which the coat protein has a beta-structure and the tryptophan indole rings do not experience the motion of the lipids in the bilayer because of protein-protein aggregation.