Repair and remodelling in the shells of the limpet Patella vulgata.

Limpets and other molluscs rely on shells to protect them from physical damage, predation, dehydration, etc. If the shell becomes damaged, this may significantly impair its function. In this work, experiments were carried out to investigate the effect of damage on the strength of shells of the common limpet (Patella vulgata) and their ability to repair this damage effectively. Shells were damaged in three ways: (i) low-energy impacts; (ii) abrasion of the outer layer; and (iii) creation of a small hole in the apex of the shell. Shells were left to repair for several time periods (0, 10, 30 and 60 days). The mechanical strength was evaluated by impacting the shells with a weight dropped from a known height. The damage reduced the strength (defined as impact energy to failure) by 50-70% depending on damage type. After 60 days, limpets in all three groups had repaired their shells significantly, bringing their strength to 79-91% of the control value (in each case, samples were statistically indistinguishable from their control counterparts). Measurements of the thickness of the shell at the apex suggest that the main effect of low-energy impact and abrasion is reduction in thickness, which correlates linearly with the impact energy needed for failure. The method of repair is believed to be by the growth of fresh shell material on the inside of the shell, though we could not identify this new material specifically. Even after 60 days, the shells were still statistically thinner than the controls. Consequently, there may be some other strengthening mechanism at work. This work has demonstrated the remarkable ability of limpets to detect the mechanical weakening of their shells caused by relatively subtle forms of damage and to take appropriate action to restore shell strength.

Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter.

The bacterial outer membrane transporter for vitamin B(12), BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane protein TonB. This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box. In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P. In the wild-type protein, the Ton box is folded into the barrel of the transporter. The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm. In the wild-type protein, vitamin B(12) induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box. Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique. The failure of these mutants to execute the B(12) transport cycle may be a result of the altered conformation of the Ton box.

Location and dynamics of basic peptides at the membrane interface: electron paramagnetic resonance spectroscopy of tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid-labeled peptides.

The attractive interaction between basic protein domains and membranes containing acidic lipids is critical to the membrane attachment of many proteins involved in cell signaling. In this study, a series of charged model peptides containing lysine, phenylalanine, and the spin-labeled amino acid tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) were synthesized, and electron paramagnetic resonance (EPR) spectroscopy was used to determine their position on the membrane interface and free energy of binding. When membrane-bound, peptides containing only lysine and TOAC assume an equilibrium position within the aqueous double layer at a distance of approximately 5 A from the membrane interface, a result that is consistent with recent computational work. Substitution of two or more lysine residues by phenylalanine dramatically slows the backbone diffusion of these peptides and shifts their equilibrium position by 13-15 A so that the backbone lies several angstroms below the level of the lipid phosphate. These results are consistent with the hypothesis that the position and free energy of basic peptides when bound to membranes are determined by a long-range Coulombic attraction, the hydrophobic effect, and a short-range desolvation force. The differences in binding free energy within this set of charged peptides is not well accounted for by the simple addition of free energies based upon accepted side chain partition free energies, a result that appears to be in part due to differences in membrane localization of these peptides.

Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin.

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.

The secretory carrier membrane protein family: structure and membrane topology.

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.

Identifying conformational changes with site-directed spin labeling.

Site-direct spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for detecting structural changes in proteins. This review provides examples that illustrate strategies for interpreting the data in terms of specific rearrangements in secondary and tertiary structure. The changes in the mobility and solvent accessibility of the spin label side chains, and in the distances between spin labels, report (i) rigid body motions of alpha-helices and beta-strands (ii) relative movements of domains and (iii) changes in secondary structure. Such events can be monitored in the millisecond time-scale, making it possible to follow structural changes during function. There is no upper limit to the size of proteins that can be investigated, and only 50-100 picomoles of protein are required. These features make site-directed spin labeling an attractive approach for the study of structure and dynamics in a wide range of systems.

Substrate-induced exposure of an energy-coupling motif of a membrane transporter.

BtuB is an outer membrane protein responsible for the uptake of vitamin B12 by Escherichia coli. It belongs to a family of bacterial transport proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling protein TonB. Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB. In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter. Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm. This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion. In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate. These data suggest that the TonB box extends into the periplasm and interacts with TonB only in

Continuum solvent model calculations of alamethicin-membrane interactions: thermodynamic aspects.

Alamethicin is a 20-amino acid antibiotic peptide that forms voltage-gated ion channels in lipid bilayers. Here we report calculations of its association free energy with membranes. The calculations take into account the various free-energy terms that contribute to the transfer of the peptide from the aqueous phase into bilayers of different widths. The electrostatic and nonpolar contributions to the solvation free energy are calculated using continuum solvent models. The contributions from the lipid perturbation and membrane deformation effects and the entropy loss associated with peptide immobilization in the bilayer are estimated from a statistical thermodynamic model. The calculations were carried out using two classes of experimentally observed conformations, both of which are helical: the NMR and the x-ray crystal structures. Our calculations show that alamethicin is unlikely to partition into bilayers in any of the NMR conformations because they have uncompensated backbone hydrogen bonds and their association with the membrane involves a large electrostatic solvation free energy penalty. In contrast, the x-ray conformations provide enough backbone hydrogen bonds for the peptide to associate with bilayers. We tested numerous transmembrane and surface orientations of the peptide in bilayers, and our calculations indicate that the most favorable orientation is transmembrane, where the peptide protrudes approximately 4 A into the water-membrane interface, in very good agreement with electron paramagnetic resonance and oriented circular dichroism measurements. The calculations were carried out using two alamethicin isoforms: one with glutamine and the other with glutamate in the 18th position. The calculations indicate that the two isoforms have similar membrane orientations and that their insertion into the membrane is likely to involve a 2-A deformation of the bilayer, again, in good agreement with experimental data. The implications of the results for the biological function of alamethicin and its capacity to oligomerize and form ion channels are discussed.

Reconstitutive refolding of diacylglycerol kinase, an integral membrane protein.

While the formation of kinetically trapped misfolded structural states by membrane proteins is related to a number of diseases, relatively few studies of misfolded membrane proteins in their purified state have been carried out and few methods for refolding such proteins have been reported. In this paper, misfolding of the trimeric integral membrane protein diacylglycerol kinase (DAGK) is documented and a method for refolding the protein is presented; 65 single-cysteine mutants of DAGK were examined. A majority were found to have lower-than-expected activities when purified into micellar solutions, with additional losses in activity often being observed following membrane reconstitution. A variety of evidence indicates that the low activities observed for most of these mutants results from kinetically based misfolding of the protein, with misfolding often being manifested by the formation of aberrant oligomeric states. A method referred to as "reconstitutive refolding" for correcting misfolded DAGK is presented. This method is based upon reconstituting DAGK into multilamellar POPC vesicles by dialyzing the detergent dodecylphosphocholine out of mixed micellar mixtures. For 55 of the 65 mutants tested, there was a gain of DAGK activity during reconstitutive refolding. In 33 of these cases, the gain in activity was greater than 2-fold. The refolding results for cysteine replacement mutants at DAGK sites known to be highly conserved provide teleological insight into whether sites are conserved, because they are critical for catalysis, for maintenance of the proper folding pathway, or for some other reason.

Interactions controlling the membrane binding of basic protein domains: phenylalanine and the attachment of the myristoylated alanine-rich C-kinase substrate protein to interfaces.

Basic residues are known to play a critical role in the attachment of protein domains to membrane interfaces. Many of these domains also contain hydrophobic residues that may alter the binding and the position of the domain on the interface. In the present study, the role of phenylanine in determining the membrane position, dynamics and free energy of a peptide derived from the effector domain of the myristoylated alanine-rich C-kinase substrate (MARCKS) protein was examined. Deuterium NMR in membranes containing phosphatidylcholine (PC) and phosphatidylserine (PS) indicates that this peptide, MARCKS(151-175), partially penetrates the membrane interface when bound and alters the effective charge density on the membrane interface by approximately 2 charges per bound peptide. However, a derivative of this peptide in which the five phenylalanines are replaced by alanine, MARCKS-Ala, does not penetrate the interface when membrane-bound. This result was confirmed by depth measurements by electron paramagnetic resonance spectroscopy on several spin-labeled derivatives of the Phe-less derivative. In contrast to nitroxides on MARCKS(151-175), nitroxides on the derivative lacking Phe do not reside within the bilayer but are in the aqueous phase when the peptide is bound to the membrane. The Phe to Ala substitutions shift the position of the labeled side chains by approximately 10-15 A. The side-chain dynamics of MARCKS-Ala are strongly influenced by membrane charge density and indicate that this peptide is drawn closer to the membrane interface at higher charge densities. As expected, MARCKS-Ala binds more weakly to membranes composed of PS/PC (1:9) than does the native MARCKS peptide; however, each phenylalanine contributes only 0.2 kcal/mol to the binding energy difference, far less than the 1.3 kcal/mol expected for the binding of phenylalanine to the membrane interface. This energetic discrepancy and the differences in membrane position of these peptides can be accounted for by a dehydration energy that is encountered as the peptide approaches the membrane interface. This energy likely includes a Born repulsion acting between the charged peptide and the low dielectric membrane interior. The interplay between the long-range attractive Coulombic force, the short-range repulsive force and the hydrophobic effect controls the position and energetics of protein domains on acidic membrane interfaces.

Distance estimates from paramagnetic enhancements of nuclear relaxation in linear and flexible model peptides.

The distance dependence of electron-nuclear dipole-dipole coupling was tested using a series of poly-L-proline based peptides of different length. The poly-proline based peptides were synthesized with a nitroxide spin label on the N-terminus and a tryptophan on the C-terminus, and paramagnetic enhancements of nuclear spin-lattice relaxation rates were measured for the aromatic protons on the tryptophan as a function of the number of proline spacers in the sequence. As expected, paramagnetic enhancements decrease with distance, but the distances deduced from the NMR relaxation rates were shorter than expected for every peptide studied compared to a rigid linear poly-L-proline type II helix structure. Calculations of cross-relaxation rates indicate that this difference is not the result of spin-diffusion or the creation of a spin-temperature gradient in the proton spins caused by the nitroxide. Molecular dynamics simulations were used to estimate dynamically averaged value of (2). These weighted average distances were close to the experimentally determined distances, and suggest that molecular motion may account for differences between the rigid linear models and the distances implied by the NMR relaxation data. A poly-L-prolone peptide synthesized with a central glycine hinge showed dramatic relaxation rate enhancements compared to the peptide of the same length lacking the hinge. Molecular dynamics simulations for the hinged peptide support the notion that the NMR data is a representation of the weighted average distance, which in this case is much shorter than that expected for an extended conformation. These results demonstrate that intermoment distances based on NMR relaxation rates provide a sensitive indicator of intramolecular motions.

Correlation between the free energy of a channel-forming voltage-gated peptide and the spontaneous curvature of bilayer lipids.

The aqueous-membrane partitioning of alamethicin, a voltage-gated channel-forming peptide, was measured as a function of the membrane spontaneous curvature. EPR spectroscopy was used to measure the partitioning of the peptide in lipid compositions formed from dioleoylphosphatidylcholine (DOPC) and varied percentages of dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine-N-methyl (DOPE-Me), or dioleoylphosphatidylethanolamine-N,N-dimethyl (DOPE-Me2). When the mole fraction of DOPE in mixtures of DOPC/DOPE is increased the binding of alamethicin decreases, and the increase in binding free energy is found to be linearly dependent upon the mole fraction of DOPE in the mixture. Addition of DOPE-Me or DOPE-Me2 also increases the binding free energy, except that the effect is reduced relative to that of DOPE. The free-energy increase per mole fraction of DOPE was found to be 1400 cal/mol, whereas for DOPE-Me and DOPE-Me2 the free-energy changes were 980 and 630 cal/mol, respectively. When the free-energy changes for alamethicin binding are compared with the previously determined spontaneous curvatures for mixtures of DOPC/DOPE and DOPC/DOPE-Me, the free energy of binding is found to be linearly dependent upon the spontaneous curvature of the bilayer lipids. The effects of membrane lipid unsaturation on the partitioning of alamethicin were also measured and are qualitatively consistent with this conclusion. The sensitivity to spontaneous curvature and the cooperativity that is seen in the binding curves for alamethicin are postulated to be a result of a localized thinning of the bilayer promoted by this peptide.

The role of proline and glycine in determining the backbone flexibility of a channel-forming peptide.

Alamethicin is a helical 20-amino acid voltage-gated channel-forming peptide, which is known to exhibit segmental flexibility in solution along its backbone near alpha-methylalanine (MeA)-10 and Gly-11. In an alpha-helical configuration, MeA at position 10 would normally hydrogen-bond with position 14, but the presence of proline at this position prevents the formation of this interhelical hydrogen bond. To determine whether the presence of proline at position 14 contributes to the flexibility of this helix, two analogs of alamethicin were synthesized, one with proline 14 replaced by alanine and another with both proline 14 and glycine 11 replaced by alanine. The C-termini of these peptides were derivatized with a proxyl nitroxide, and paramagnetic enhancements produced by the nitroxide on the Calpha protons were used to estimate r-6 weighted distances between the nitroxide and the backbone protons. When compared to native alamethicin, the analog lacking proline 14 exhibited similar C-terminal to Calpha proton distances, indicating that substitution of proline alone does not alter the flexibility of this helix; however, the subsequent removal of glycine 11 resulted in a significant increase in the averaged distances between the C- and N-termini. Thus, the G-X-X-P motif found in alamethicin appears to be largely responsible for mediating high-amplitude bending motions that have been observed in the central helical domain of alamethicin in methanol. To determine whether these substitutions alter the channel behavior of alamethicin, the macroscopic and single-channel currents produced by these analogs were compared. Although the substitution of the G-X-X-P motif produces channels with altered characteristics, this motif is not essential to achieve voltage-dependent gating or alamethicin-like behavior.

Structural features that modulate the transmembrane migration of a hydrophobic peptide in lipid vesicles.

Two approaches employing nuclear magnetic resonance (NMR) were used to investigate the transmembrane migration rate of the C-terminal end of native alamethicin and a more hydrophobic analog called L1. Native alamethicin exhibits a very slow transmembrane migration rate when bound to phosphatidylcholine vesicles, which is no greater than 1 x 10(-4) min(-1). This rate is much slower than expected, based on the hydrophobic partition energies of the amino acid side chains and the backbone of the exposed C-terminal end of alamethicin. The alamethicin analog L1 exhibits crossing rates that are at least 1000 times faster than that of native alamethicin. A comparison of the equilibrium positions of these two peptides shows that L1 sits approximately 3-4 A deeper in the membrane than does native alamethicin (Barranger-Mathys and Cafiso. 1996. Biochemistry. 35:489). The slow rate of alamethicin crossing can be explained if the peptide helix is irregular at its C-terminus and hydrogen bonded to solvent or lipid. We postulate that L1 does not experience as large a barrier to transport because its C-terminus is already buried within the membrane interface. This difference is most easily explained by conformational differences between L1 and alamethicin rather than differences in hydrophobicity. The results obtained here demonstrate that side-chain hydrophobicity alone cannot account for the energy barriers to peptide and protein transport across membranes.

Influence of lipid on the structure and phosphorylation of protein kinase C alpha substrate peptides.

The structure and phosphorylation of two protein kinase C (PKC) alpha substrate peptides were investigated in varying lipid systems using enzyme activity assays and circular dichroism (CD) spectroscopy. The alpha-peptide, which exhibits the typical PKC alpha substrate motif and is based on the pseudosubstrate region of PKCalpha, was phosphorylated to a similar extent in bovine brain phosphatidylserine vesicles or diheptanoylphosphatidylcholine (PC7) micelles (both with 5 mol % 1,2-dioleoyl-sn-glycerol), whereas neuromodulin (NM)-peptide, which does not exhibit this motif by virtue of its primary structure, was phosphorylated to a much lesser extent in the PC7 micellar system. CD spectra of the peptides indicated that NM-peptide underwent a dramatic structural change in the presence of dimyristoylphosphatidylserine (DMPS) vesicles, whereas spectra acquired in PC7 micelles were similar to those acquired in buffer alone. No significant structural change was observed in the alpha-peptide in the presence of either lipid. PKC activity assays conducted with a series of NM-peptides successively substituted with nitroxide spin labels at each residue position suggested that several residues distal to the phosphorylation site are necessary for substrate recognition. The effect of these substitutions is not consistent with the binding of the NM-peptide to PKC in an extended structure, but is consistent with the binding of this peptide in a helical conformation. Furthermore, the docking of a helical NM-peptide to the substrate binding site of PKC suggests that the interaction is energetically feasible. These results suggest that PKC may recognize some non-linear substrate motifs and that lipid binding may convert a protein into a better PKC substrate.

Structure and position of the N-terminal membrane-binding domain of pp60src at the membrane interface.

Hydrophobic and electrostatic interactions between the acylated N-terminal end of Src and lipid bilayers are responsible for the attachment of this nonreceptor tyrosine kinase to the membrane-solution interface. To investigate the structure and dynamics of this domain at the membrane interface, a series of peptides based upon the N-terminal end of pp60src, myr-src(2-16), was synthesized with single-site cysteine substitutions and derivatized with a sulfhydryl-reactive proxyl nitroxide. The EPR line shapes and mobility of these peptides when bound to the membrane interface were consistent with an extended peptide conformation, and no evidence was found for either a helical or sheet structure. Line shapes on the myristoylated N-terminal end indicate that this segment is more restricted in its motion than at the C-terminus. Although the membrane affinity of this peptide is much stronger in the presence of acidic lipid, EPR line shapes were not strongly affected by the presence of acidic lipid. An EPR power saturation technique was used to provide information on the position of nitroxides from the interface for the membrane-bound peptide. When membrane bound, labeled side chains at the N-terminal end of the peptide were found to lie in the aqueous phase near the membrane interface; however, for the C-terminal half of the peptide, residues were further off the membrane and were 10-15 A from the interface. Peptides derived from the membrane and calmodulin binding domains of the myristoylated alanine-rich C kinase substrate and neuromodulin were previously found to be in extended conformations; however, side chains for these peptides penetrated the membrane-solution interface. We speculate that the relatively polar character of the N-terminal segment of Src and a Born repulsion energy prevent this peptide from penetrating into the membrane interface when membrane bound.

Dipole potentials and spontaneous curvature: membrane properties that could mediate anesthesia.

General anesthetics alter both the membrane dipole potential and the membrane spontaneous curvature, two membrane properties that are likely to have a significant effect on membrane protein function. The dipole potential is a large hydrocarbon positive potential that appears to arise from the lipid carbonyl groups and/or water at the membrane-solution interface. Anesthetics reduce the magnitude of the membrane dipole potential at clinical levels of anesthetics, while non-anesthetics do not, and these changes in potential could modulate conformational transitions in membrane proteins that are electrically active. When the membrane distribution of anesthetic versus non anesthetic compounds is examined, anesthetics exhibit a preference for the membrane interface, whereas non-anesthetic compounds reside within the membrane hydrocarbon core. The preferential localization of anesthetics within the interface accounts for their effect on the membrane dipole potential, and may also serve to alter the membrane spontaneous curvature or lateral stress through the bilayer.

Estimating the electrostatic potential at the acetylcholine receptor agonist site using power saturation EPR.

Continuous wave EPR power saturation was used to measure electrostatic potentials at spin-labeled sites. Membrane surface potentials were estimated by power saturating the EPR spectrum of a membrane bound 14N spin-labeled amphiphile in the presence of a neutral or positively charged 15N labeled aqueous spin probe. The potentials that are measured are in good agreement with other probe measurements and with the predictions of the Gouy-Chapman-Stern theory, indicating that this is a valid approach to determine electrostatic potentials. A spin-labeled affinity probe based on maleimidobenzyltrimethylammonium was synthesized and could be derivatized to a sulfhydryl near either agonist site on the nicotinic acetylcholine receptor. The amplitudes of motion of the spin-probe on the ns time scale are significantly different when the two labeled sites are compared, and the probe is more restricted in its motion when attached to the more easily labeled site. When attached to this agonist site, power saturation EPR yields an electrostatic potential of -15 mV. Two other sulfhydryl-specific probes were used to label this site in reconstituted receptor containing membranes. These probes show less contact with the receptor and reduced electrostatic potentials, indicating that there is a strong spatial dependence to the potential at the agonist site. This work demonstrates that power saturation EPR provides a general method that can be used to estimate electrostatic potentials at any specifically spin-labeled macromolecular site.

Water translational motion at the bilayer interface: an NMR relaxation dispersion measurement.

Nuclear magnetic relaxation rates for water protons in aqueous palmitoyloleoylphosphatidylcholine vesicle suspensions containing different nitroxide free radical spin labels are reported as a function of magnetic field strength corresponding to proton Larmor frequencies from 10 kHz to 30 MHz. Under these conditions the water proton relaxation rate is determined by the magnetic coupling between the water protons and the paramagnetic nitroxide fixed on the phospholipid. This coupling is made time-dependent by the relative translational motion of the water proton spins past the nitroxide radical. Using theories developed by Freed and others, we interpret the NMR relaxation data in terms of localized water translational motion and find that the translational diffusion constant for water within approximately 10 A of the phospholipid surface is 6 x 10(-10) m2 s(-1) at 298 K. Similar results are obtained for three different nitroxide labels positioned at different points on the lipid. The diffusion is a thermally activated process with an activation energy only slightly higher than that for bulk water.

Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin.

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, kon = 10(11) M-1 s-1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, kon is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (koff) is approximately 10 s-1 (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca2+-calmodulin (Ca2+.CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 micrM Ca2+.CaM. Our results suggest that Ca2+.CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4, 5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).