Region Of Lipid Bilayer Research Articles

Melittin-induced alterations in dynamic properties of human red blood cell membranes

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10 −10 mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10 −10 mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10 −10 mol/mg (10 −4 mol melittin per mole of membrane phospholipid). The decrease in the ratio h w h s of maleimide and the rise in relative rotational correlation time ( τ c) of iodoacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.

Cholesterol modulation of lipid-protein interactions in liver microsomal membrane: a spin label study.

ESR spectra of spin probes were used to monitor lipid-protein interactions in native and cholesterol-enriched microsomal membranes. In both systems composite spectra were obtained, one characteristic of bulk bilayer organization and another due to a motionally restricted population, which was ascribed to lipids in a protein microenvironment. Computer spectral subtractions revealed that cholesterol modulates the order/mobility of both populations in opposite ways, i.e., while the lipid bilayer region gives rise to more anisotropic spectra upon cholesterol enrichment, the spectra of the motionally restricted population become indicative of increased mobility and/or decreased order. These events were evidenced by measurement of both effective order parameters and correlation times. The percentages of the motionally restricted component were invariant in native and cholesterol-enriched microsomes. Variable temperature studies also indicated a lack of variation of the percentages of both spectral components, suggesting that the motionally restricted one was not due to protein aggregation. The results correlate well with the effect of cholesterol enrichment on membrane-bound enzyme kinetics and on the behavior of fluorescent probes [Castuma & Brenner (1986) Biochemistry 25, 4733-4738]. Several hypothesis are put forward to explain the molecular mechanism of the cholesterol-induced spectral changes.

Aspects of the transdermal delivery of prostaglandins

The skin penetration of prostaglandins E 1, E 2, F 1α and F 2α has been studied in vitro in human skin. The absorption rates are influenced by the presence of the enhancers Azone ® and Transcutol. The results indicated different potential interactions which were structure related. These were further investigated using both bilayers and monolayers of dipalmitoylphosphatidylcholine (DPPC) to mimic stratum corneum lipids. Pretreatment of skin with both Azone and Transcutol was found to facilitate drug fluxes. This was especially the case with the 2-series prostaglandins where, both with, and without enhancer pretreatment, the actual amounts penetrating were greater than for the 1-series. A bilayer model (DPPC multilamellar vesicles) indicated that the 2-series compounds have a greater disruptive effect on structured lipids than their 1-series counterparts. Monolayer experiments demonstrated the mechanism of disruption to he non-penetrative, implying interaction between the 2-series prostaglandins and the DPPC head-group region. The major barrier to prostaglandin penetration appears to reside in the lipid region of the stratum corneum bilayers and the 2-series prostaglandins may interact with polar head-groups in this region in such a way as to ease their passage through them. The reason why the 1-series proslaglandins are absorbed comparatively poorly probably lies in their lack of compatibility with the skin membrane constituents and their consequent inability to interact with and destabilize the head-group regions of the stratum corneum lipid bilayers.

Membrane fusion induced by mutual interaction of the two charge-reversed amphiphilic peptides at neutral pH

An anionic amphiphilic peptide and the charge-reversed cationic peptide are synthesized. They contain 20 amino acids with the same sequence except for 5 Glu residues for the anionic versus 5 Lys residues for the cationic peptides. Fusion of egg phosphatidylcholine large unilamellar vesicles is assayed with the fluorescent probes by the lipid mixing and the internal content mixing at neutral pH. The peptide mixture causes a rapid and efficient membrane fusion, in spite of no fusions with each peptide by itself. Each peptide takes nearly random coils with a small amount of helix, but the peptide mixture has an ordered helical structure. The equimolar peptide mixture forms a much more hydrophobic complex than those of different molar ratios of peptides and also that of each peptide itself. The equimolar peptide mixture causes the most efficient fusion. Preincubations of two peptides before addition to vesicles cause the slower rates of fusion. The fusion is greatly reduced at higher ionic strength and nearly zero at 800 mM NaCl and 40 mM sodium phosphate. Each peptide and the peptide mixture show the same alpha-helical structure, interact with vesicles, but do not induce fusion at higher ionic strengths. These results suggest that the two peptides interact mutually through the electrostatic Coulombic interaction between the charged groups. The electrically neutralized hydrophobic complex aggregates the separate vesicles together and interacts with the hydrocarbon region of lipid bilayers to cause fusion.

Ionizing radiation-induced structural modification of human red blood cells

The effect of gamma radiation on red blood cells have been examined using a spin labeling method. For this purpose two spin labels were used to monitor membrane fluidity: methyl 5-doxylpalmitate (Met 5-DP) and methyl 12-doxylstearate (Met 12-DS). The irradiation of red cells with the doses of 200 and 500 Gy caused decrease of microviscosity in certain regions of lipid bilayer (as indicated by Met 5-DP and Met 12-DS spectra) but did not affect lipid order parameter. The behavior of two other spin labels, maleimide(4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl) indicated: 1) conformational changes of membrane proteins, 2) modification of cell internal peptides and proteins, 3) decreased internal viscosity of red blood cells.

Alloxan-induced alterations in composition and dynamics of red blood cell membranes: I. Effect of alloxan on intact red blood cells and isolated erythrocyte membranes

Changes of dynamics and chemical composition in membranes of intact red blood cells and isolated erythrocyte membranes treated with alloxan were investigated in order to assess whether alloxan-induced generation of active forms of oxygen may be critical for erythrocyte destroying. In vitro incubation of native red blood cells or prepared erythrocyte membrane ghosts with various concentrations of alloxan gave rise both to levels of membrane TBA-reacting substance and lipid membrane micro-viscosity both in the deeper and surface regions of lipid bilayer, as evidenced by fluorescence polarization technique. The amount of membrane phospholipid decreased upon alloxan action and that of membrane cholesterol remained rather unchangeable, thus resulting in significant elevation of membrane cholesterol : phospholipid (C : PL) ratio. Both time course and concentration effect of alloxan were found to change exponentially with the different rates of the reaction. There was a linear correlation between 1,6-diphenylhexatriene-1,3,5 (DPH) and 1-anilinonaphthalene-8-sulfonate (ANS) anisotropy coefficients and C : PL ratio (respectively r = 0.697 and r = 0.580) as well as TEARS levels ( r = 0.386 for r DPH and r = 0.324 for r ANS), thus implying the possible effect of membrane dialdehydes on bilayer components immobilization. Regression coefficients significance testing showed reaction rates ofTBARS and C:PL changes to be significantly parallel, contrary to those of fluorescence anisotropy coefficients assessing considerably slower dynamics of alloxan-induced changes. The relevance of changes induced by alloxan in isolated erythrocyte ghosts and intact red blood cells and the compatibility of the present results with several previous studies support the widespreading idea pointing the cell membrane as a main target of damage during alloxan action.

Phase behaviour of lipid X

The phase behaviour of aqueous dispersions of lipid X, a precursor of bacterial lipopolysaccharides has been investigated by a variety of physico-chemical techniques. The results are consistent with the presence of disk-shaped micelles with an average diameter of 13 ± 1.8 nm. The critical micellar concentration in water and physiological saline is 4 × 10 −5 M. Consistent with the formation of micelles in water and physiological saline is the finding that lipid X is in the liquid-crystalline state at temperatures higher than 0°C. The packing and the dynamics of lipid X are characteristic of micelles. Close to the polar group the hydrocarbon chains are significantly more mobile and disordered than in the corresponding region of lipid bilayers. From monolayer studies an estimate of the molecular area of lipid X is derived; under physiological conditions the area/molecule is about 0.50 nm 2 at 30 mN/m indicating that lipid X has a wedge-like shape. The two p K values of the primary phosphate group of lipid X are p K 1 ≈ 1.3 and p K 2 = 8.2. At pH values < 7, the area/molecule decreases, i.e. the packing of the lipid X molecules becomes tighter, and there is also a decrease in the solubility of lipid X. As is characteristic of charged lipids, the state of aggregation (phase behaviour) of lipid X depends on pH, the ionic strength and the nature of the counterion.

Interaction of Oxygen Carrying Microparticles with Blood Components and Clearance from Blood Stream

The stable microparticles (polymeric liposomes) having a synthetic heme-imidazole complex and carrying oxygen were studied in the interaction with blood components in vitro. They did not induce hemolysis, platelet aggregation and plasma coagulation directly and were stable against the hydrolysis by phospholipases. Intravenous injection of the 14C-labeled particles into rats indicated the half-disappearance time from the blood stream of ca. 14 hr. Their tissue distribution was analyzed with time by autoradiography.The authors have demonstrated that a stable synthetic oxygen carrier can be prepared by embedding a synthetic heme complex into the hydrophobic region of lipid bilayers of liposomes (1–4). By the use of polymeric liposomes (5), we have succeeded in preparing a stable solution of high heme concentration (6). The new oxygen-carrying aqueous solution had physical properties similar to those of whole blood. To elucidate the further possibility of the micro-particles as red blood cell models, the inte...

Effect of proteolipid protein on central nervous system myelin membrane fluidity

The effect which intrinsic (proteolipid) protein has on fluidity of central nervous system myelin membrane was measured through differences in temperature-dependent anisotropy of the lipid-soluble fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in multilamellar vesicles (MLV) prepared from total myelin lipids in the presence and absence of proteolipid protein. Very little difference was observed in the anisotropies of DPH incorporated into intact myelin membrane vesicles compared with MLV reconstituted from total myelin lipid plus proteolipid protein but excluding myelin basic protein. In contrast, a significant decrease (P < 0.01) in anisotropy was observed when MLV prepared from total myelin lipids depleted of proteolipid protein were compared with vesicles containing proteolipid protein. Given the different distributions of myelin basic protein and proteolipid protein suggested by freeze-fracture, neutron and X-ray diffraction studies, and the fact that the hydrophobic DPH probe is known to distribute in the non-polar regions of lipid bilayers, we interpret the marked decrease in anisotropy when proteolipid protein is excluded from MLV to suggest that at least part of the proteolipid is distributed in the hydrocarbon region of the MLV. These findings are consistent with the earlier physical studies and recent postulations that extensive hydrophobic segments exist in proteolipid protein and that these hydrophobic segments are buried in the myelin lipid bilayer and alternate with hydrophilic extra-membrane segments.

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 Å resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 Å resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.

Giant planar lipid bilayer. III. Maxwell—Wagner impedance dispersion and anesthetic effects upon interfacial capacitance

This report describes a facile linear method for estimating the capacitance of the polar head region of planar lipid bilayers from the dependence of the impedance to the applied frequency at an intermediate range. Planar lipid bilayers with a large area (200 mm 2) were formed in 30 m M NiCl 2 aqueous solution from oleylamine. The capacitance and conductance of the total system, including the water, unstirred layer and membrane phases, were measured in the frequency range between 1 and 10 kHz. In this frequency range, a linear relationship exists between the overall (hydrophobic core plus polar head region) capacitance and conductance. By extrapolating the linear plot between the capacitance and conductance to zero conductance, the capacitance of the overall membrane was estimated. The capacitance of the hydrocarbon region of the membrane was estimated by the do transient method. From the difference in the two sets of data, it is possible to estimate the capacitance of the interfacial polar region. According to the increase of the bulk halothane concentrations, the capacitance of the polar region decreased asymptotically, approaching a limiting value. The decrease was monophasic. On the other hand, the core capacitance increased in a biphasic fashion, as reported previously by T. Yoshida, T. Mori, and I. Ueda ( J. Colloid Interface Sci. 96, 39, 1983). The initial increase in the core capacitance was attributed to the decrease in the membrane thickness, whereas the secondary rise was attributed to anesthetic penetration into the membrane core. Because the relative electrical permittivity of anesthetics is intermediate between those of the hydrocarbon core and the interface, the anesthetic adsorption to the polar head decreases the mean relative permittivity of this region and decreases the capacitance, whereas penetration into the lipid core increases the core capacitance. The present result shows that the secondary rise of the core capacitance occurred only after polar region capacitance reached a limiting value. Halothane apparently adsorbs to the polar head region at near clincal concentrations. Penetration into the liquid core did not occur until the anesthetic molecules saturated the interfacial binding sites.

Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels

Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog rod outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodopsin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006–5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes.

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Mg2+-mediated change in lipid fluidity enhances the reconstituted H+-ATPase activity.

The results by using ANS fluorescent probe and spin labels 5-NS show that the fluidity of L. (H+-ATPase) +Mg2+ (H+-ATPase from pig heart mitochondria reconstituted in the presence of Mg2+) is less than that of L. (H+-ATPase) -Mg2+ (proteoliposome reconstituted in the absence of Mg2+). But no significant difference in fluidity has been observed when both reconstituted systems were monitored by using spin labels 12-NS and 16-NS. This indicates that Mg2+ may cause changes in fluidity of the lipid molecules near the surfaces of the bilayers, but does not affect significantly the fluidity of the deeper layer of the reconstituted system. It is tentatively supposed that in the presence of Mg2+, enhancement of activities of reconstituted H+-ATPase may be due to the Mg2+-mediated change in physical state of the lipids in the more superficial region of lipid bilayers so as to ensure a suitable conformation of ATPase complex, thereby possessing higher activity.

Concentration of oxygen in lipid bilayers using a spin-label method

The concentration of oxygen in the hydrocarbon region of lipid bilayer has been determined using a novel electron spin resonance (ESR) nitroxide-radical spin-probe method. For dimyristoylphosphatidylcholine (DMPC), the partition coefficient above the main transition temperature is approximately 3. Rapid decrease to 0.2 occurs below the pretransition temperature indicating exclusion of oxygen in the crystalline phase. The differences of molar free energy, enthalpy, and entropy of mixing between water and lipid have been determined for each phase.

Changes in the organization of the outer membrane of Proteus mirabilis during swarming: freeze-fracture structure and membrane fluidity analysis.

Freeze-fracture studies of short, nonswarming Proteus mirabilis revealed the characteristic gram-negative profile of fractured inner membrane with densely packed particles and sectioned outer membrane with little or no fracture plane. Long swarming cells, however, fractured easily along both the inner membrane and a second membrane, probably the outer membrane. The inner membrane had a typical profile, whereas the outer membrane had fewer but more prominent particles. Isolation and purification of the inner and outer membranes of the short and long bacteria and examination of them with electron paramagnetic resonance measurements after spinlabeling supported the above observations. The outer membrane of swarmer cells allowed higher mobility of the spin label than did the outer membrane of the nonswarming short cells, which showed a typical rigid profile. These results suggest that regions of lipid bilayer appear in the outer membrane during swarmer formation. Previous observation of the behavior and biochemistry of P. mirabilis during swarming are discussed in light of these results.

Lectin‐induced rearrangement of an immature hematopoietic cell surface marker

Experiments were carried out to examine the possible physiological role of disordered membrane domains in hematopoietic cell surface differentiation. The hematopoietic stem cell line 416B has been shown to bind the dye merocyanine 540 (MC540), a fluorescent probe which may be specific for disordered regions of lipid bilayers (Williamson et al., 1981). The surface receptors for the lectins Concanavalin A (Con A) and wheat germ agglutinin (WGA) exhibit patchy distributions on the surface of 416B cells which correspond to the distribution of MC540 binding regions. Appropriate incubation of these cells with either of the two lectins results in the induced formation of a cap. The binding regions for MC540 and the receptors for the other lectin become localized to the same region of the membrane by this process. Such coordinated rearrangements of surface glycoproteins associated with disordered lipid domains may play a role in the cocapping of disparate surface molecules (Raz and Bucana, 1980) or in differentiation-related cell surface rearrangements (Schlegel et al., 1980).

5-(dimethylamino)naphthalene-1-sulfonic acid, a fluorescent probe of the medium chain fatty acid binding site of serum albumin.

Reversible binding of 5-(dimethylamino)-naphthalene-1-sulfonic acid (DNS) to human and bovine serum albumin has been monitored by changes in fluorescence intensity, wavelength maxima, and polarization. DNS has only one major binding site (ka = 5 x 10(6)) and one minor site (ka = 3 x 10(5)) on these proteins. The probe is competitively displaced from its high-affinity site by medium chain fatty acids and by N-acetyl-L-tryptophan. The binding site for DNS is highly hydrophobic and is considerably less polar than the hydrocarbon region of lipid bilayers. Resonance energy transfer indicates that the binding site is located 20.7 +/- 2 A from the single tryptophan residue of human serum albumin.

91] Fluorescent probe: Diphenylhexatriene

Publisher Summary This chapter discusses the fluorescent probe—diphenylhexatriene. Depolarization of fluorescence has proved to be a very reliable technique with which to characterize the thermotropic and dynamic aspects of the hydrophobic regions of lipid bilayers and lipoproteins. Fluorescence analysis of dynamic systems can be performed either with time-dependent or steady-state type anisotropy measurements. The former approach can yield information concerning the heterogeneity of the lifetime of the fluorophore in the system, allowing in some cases a resolution of fluorophore subpopulations. This technique also allows an evaluation of the hindrance of the probe motion because of the degree of order of the phospholipid acyl side chains. Fluorescence polarization is measured by exciting the fluorophore with monochromatic light through a polarizer whose polarization axis is oriented vertically to the light path and the emission intensity is detected through an analyzer, whose polarization axis is oriented sequentially parallel to and perpendicular to the polarization axis of the exciting light.

Interactions of wild-type and mutant M protein of vesicular stomatitis virus with viral nucleocapsid and envelope in intact virions. Evidence from [125I]iodonaphthyl azide labeling and specific cross-linking.

Four different temperature-sensitive M protein mutants (tsM) of vesicular stomatitis virus (VSV) were characterized with regard to the association of the mutated M protein either with nucleocapsids or with membranes in the intact virions. Virions were labeled with the photoreactive hydrophobic probe [125I]iodonaphthyl azide (INA) to assess interactions between viral proteins and the lipid envelope. In wild type (wt) virions, the three major structural proteins--G, M, and N--were labeled in the ratio ca. 1.0:0.4:0.2. INA labeled only the membrane-associated peptide of G protein, both in the intact virion and in reconstituted G protein--viral lipid vesicles, demonstrating the specificity of INA for lipid bilayer regions. Labeling of tsM virions with INA resulted in a 2--3-fold greater incorporation into M protein than was found for wt virions, suggesting increased M--membrane associations in the mutant virions. Temperature-stable revertants from tsM possessed wt labeling characteristics. Interaction of the M protein with nucleocapsids was assessed from the abundance of disulfide-linked M--N complexes found after disruption of the virions by sodium dodecyl sulfate solution under nonreducing conditions. The abundance of such complexes was 30--80% less from tsM virions than from wt virions, suggesting decreased M--nucleocapsid interactions in tsM virions. Temperature-stable revertants from tsM resembled wt in the abundance of M--N complex formed. We conclude that the mutations alter M protein in such a way as simultaneously to increase its association with membrane and to decrease its affinity for nucleocapsids in the intact virion.