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Biomedical subjects

D S Cafiso

Publications and source records attributed to D S Cafiso.

At least 37 records · Page 2Linked to original sources
PubMed1997

Contrasting membrane localization and behavior of halogenated cyclobutanes that follow or violate the Meyer-Overton hypothesis of general anesthetic potency.

The membrane localization and properties of two halogenated cyclobutanes were examined using 2H and 19F NMR. The common predictors of potency indicate that these two compounds will have anesthetic activity; however, 1,2-dichlorohexafluorocyclobutane (c(CCIFCCIFCF2CF2)) is not an effective anesthetic, whereas 1-chloro-1,2,2-trifluorocyclobutane (c(CCIFCF2CH2CH2)) is an effective general anesthetic. Using 2H NMR, the effect of these compounds on the acyl chain packing in palmitoyl (d31) oleoylphosphatidylcholine membranes was examined. The addition of the anesthetic c(CCIFCF2CH2CH2) results in small increases in the segmental order near the headgroup, whereas segments deeper in the bilayer show decreases in order. These results are consistent with those obtained previously for halothane, isoflurane, and enflurane. On the addition of the nonanesthetic c(CCIFCCIFCF2CF2), the segmental order in vitually unchanged, except for a slightly changed order near the segents 10-12 of the palmitoyl chains. These results, and the 19F chemical shifts, indicate that the anesthetic c(CCIFCF2CH2CH2) exhibits a preference for the membrane interface, as do the other general anesthetics, whereas the nonanesthetic c(CCIFCIFCF2CF2) resides within the membrane hydrocarbon core. The compound c(CCIFCCIFCF2CF2) and other nonanesthetic halocarbons have lower molecular dipole moments compared to effective anesthetic halocarbons, which may account for their altered distribution within the membrane. These data strongly suggest that preferential localization of a halocarbon within the membrane interface is a predictor of anesthetic potency. Furthermore, the data indicate that the properties and forces in the membrane interface deserve consideration as mediators of anesthetic activity.

Anesthetics↗
PubMed1996

Defining protein-protein interactions using site-directed spin-labeling: the binding of protein kinase C substrates to calmodulin.

EPR spectroscopy was used to examine protein-protein interactions between calmodulin and spin-labeled peptides based on the protein kinase C substrate domains of the myristoylated alanine rich C kinase substrate (MARCKS) and neuromodulin. When bound to calmodulin, the C- and N-terminal ends of a 25 residue MARCKS derived peptide exhibited large amplitude motion on the nanosecond time scale and were accessible to paramagnetic agents in aqueous solution. However, residues 5-23 were highly protected and in contact with side chains from calmodulin. These data are consistent with an alpha-helical configuration for this segment of MARCKS and with structures that have been obtained for other calmodulin-substrate complexes. For the 17 residue neuromodulin derived peptide, which is Ca2+ independent in its binding to calmodulin, oxygen collision rates demonstrate that one helical face of this peptide interacts strongly with calmodulin. The data are consistent with an interaction of this face specifically with the C-terminal lobe of calmodulin, where this lobe is either in an "open" or "semiopen" configuration. The EPR data also indicate that the N-terminal lobe of calmodulin is in contact with the peptide, but that this lobe is not as strongly associated with the peptide target. Overall, the binding pocket for neuromodulin appears to be less compact and more dynamic than that formed by MARCKS. This behavior has not previously been seen for calmodulin substrates, and it may play a role in the Ca2+ independent binding of this class of substrates. This work demonstrates the utility of EPR spectroscopy to define protein-protein interactions; in addition, oxygen collision frequencies obtained at buried sites appear to provide information on the conformational dynamics of proteins.

Amino Acid Sequence↗
PubMed1996

Solution and membrane bound structure of a peptide derived from the protein kinase C substrate domain of neuromodulin.

The solution, micelle, and membrane bound structure of a peptide based on the protein kinase C and calmodulin binding domain of neuromodulin was studied using a combination of NMR, EPR, and circular dichroism. NMR spectroscopy on this peptide indicates that there is little secondary structure in aqueous solution or detergent micelles, but that the peptide is helical in methanol. This finding is in agreement with EPR experiments utilizing double spin-labeled derivatives of the peptide as well as circular dichroism. The membrane bound structure of this peptide was investigated with EPR by synthesizing a series of spin-labeled peptides based on the protein kinase C and calmodulin binding domain of neuromodulin. These peptides exhibit no binding to neutral membranes containing phosphatidylcholine, but associate strongly with membranes containing negatively charged lipids such as phosphatidylserine. The depth of penetration of the spin label was estimated using continuous wave power-saturation EPR and demonstrates that labels at the ends of the peptide are localized slightly outside the membrane interface, but that spin labels in the central portion of the sequence are near or within the membrane interface. In addition, the peptide is in an extended structure when bound to membranes containing acidic lipid with its more hydrophobic side chains interacting with the membrane interior. The results demonstrate that the binding of these peptides to membranes is not driven by purely electrostatic interactions, but includes the interaction of hydrophobic side chains with the membrane interior.

Amino Acid Sequence↗
PubMed1996

Membrane structure of protein kinase C and calmodulin binding domain of myristoylated alanine rich C kinase substrate determined by site-directed spin labeling.

Cysteine-substituted peptides based on the membrane, calmodulin, and protein kinase C binding domain of the myristoylated alanine rich C kinase substrate (MARCKS) were synthesized and derivatized with a sulfhydryl reactive proxyl nitroxide. These spin-labeled peptides were used in combination with continuous wave power saturation electron paramagnetic resonance (EPR) spectroscopy to determine the position and structure of the peptide on membranes containing phosphatidylserine. These peptides bind at the membrane interface, with nitroxide side chains in the central and C-terminal regions lying several angstroms below the level of the head group. In contrast, the N-terminus of the peptide is extended out of the membrane interface so that the two N-terminal residues are positioned on the aqueous side of the head group. When bound to the membrane, the N-terminal segment of this peptide is sensitive to the membrane surface charge density. Higher charge densities decrease the amplitude of side chain motions at the N-terminus and bring this end of the peptide closer to the membrane interface. When the location of successive residues along the bilayer normal is compared, no helical trend is seen, and no evidence for aggregation of the peptide is found. The EPR spectra of double spin-labeled peptides also show no evidence for a helical structure. Thus, these basic peptides are in an extended configuration at the membrane interface with hydrophobic side chains oriented inward toward the membrane hydrocarbon.

Alanine↗
PubMed1996

Membrane structure of voltage-gated channel forming peptides by site-directed spin-labeling.

Three spin-labeled derivatives of the voltage-gated peptide alamethicin were prepared with nitroxides at the C-terminal phenyalaninol, and at positions 9 and 15 in the amino acid sequence. In addition, three spin-labeled derivatives of an analog of alamethicin where alpha-methylalanine residues are replaced by leucine were prepared with nitroxide labels at the same positions. Continuous wave power saturation EPR spectroscopy was used to examine the effect of molecular oxygen and water soluble paramagnetic reagents on the saturation behavior of the labeled peptides. Using the gradients of these species which exist through the membrane-solution interface, distances for these nitroxide derivatives from the membrane-solution interface were estimated. The distances show that alamethicin is inserted along the bilayer normal with the C-terminus of the peptide lying in the aqueous solution 3 or 4 A from the membrane interface. In this configuration alamethicin does not completely cross the bilayer, and the N-terminus of alamethicin is within the membrane hydrocarbon approximately 16 A from the phosphate groups on the opposing interface. The analog where leucines replace alpha-methylalanines shows a similar conformation, except that the entire peptide is translated 3-4 A deeper into the membrane than is native alamethicin. The distances that are measured for alamethicin using EPR are consistent with a linear high resolution NMR structure determined in SDS and the X-ray crystal structure. The membrane position and structure of alamethicin found here limit the likely models for voltage-gating of this peptide.

Alamethicin↗
PubMed1995

Antigen-based heteropolymers facilitate, via primate erythrocyte complement receptor type 1, rapid erythrocyte binding of an autoantibody and its clearance from the circulation in rhesus monkeys.

We investigated the feasibility of using the primate E complement receptor (CR1), in concert with Ag-based heteropolymers (AHP), as a potential therapy to remove autoantibodies from the circulation. AHP are prepared by cross-linking an anti-CR1 mAb with the acetylcholine receptor (AChR), the principal target Ag in myasthenia gravis. In vitro studies demonstrate that this methodology facilitates specific, rapid, and quantitative binding of an anti-AChR mAb to primate Es. In vivo experiments in rhesus monkeys indicate that AHP-mediated binding of an anti-AChR mAb to Es leads to the clearance of the mAb from the circulation. Once bound to the E via the AHP, the autoantibody is transported to the liver and spleen, where it is degraded without destruction of the E. It is therefore likely that the complexes of AHP and target mAb, when bound to Es, are recognized in vivo and processed by a mechanism quite similar to that which occurs when complement-opsonized immune complexes, bound to primate Es, are cleared from the circulation. It may be possible to extend and generalize this work to allow for the development of a simple, noninvasive therapy that can be made specific for the treatment of several different autoimmune diseases.

Animals↗
PubMed1995

Distribution of general anesthetics in phospholipid bilayers determined using 2H NMR and 1H-1H NOE spectroscopy.

The effect of the general anesthetics halothane, enflurane, and isoflurane on hydrocarbon chain packing in palmitoyl(d31)oleoylphosphatidylcholine membranes in the liquid crystalline phase was investigated using 2H NMR. Upon the addition of the anesthetics, the first five methylene units near the interface generally show a very small increase in segmental order, while segments deeper within the bilayer show a small decrease in segmental order. From the 2H NMR results, the chain length for the perdeuterated palmitoyl chain in the absence of anesthetic was found to be 12.35 A. Upon the addition of halothane, enflurane, or isoflurane, the acyl chain undergoes slight contractions of 0.11, 0.20, or 0.16 A, respectively, at 50 mol % anesthetic. A simple model was used to estimate the relative amounts of anesthetic located near the interface and deeper in the bilayer hydrocarbon region, and only a slight preference for an interfacial location was observed. Intermolecular 1H-1H nuclear Overhauser effects (NOEs) were measured between phospholipid and halothane protons. These NOEs are consistent with the intramembrane location of the anesthetics suggested by the 2H NMR data. In addition, the NOE data indicate that anesthetics prefer the interfacial and hydrocarbon regions of the membrane and are not found in high concentrations in the phospholipid headgroup.

Anesthetics, General↗
PubMed1995

Anesthetics reduce the magnitude of the membrane dipole potential. Measurements in lipid vesicles using voltage-sensitive spin probes.

Lipid membranes possess a large internal dipole potential that greatly exceeds the magnitude of typical transmembrane or surface potentials. The volatile general anesthetics, halothane, isoflurane and enflurane were tested by the use of positively and negatively charged hydrophobic ion spin labels in lipid bilayer vesicles for their ability to modulate the membrane dipole potential. These anesthetics decreased the binding of negatively charged hydrophobic ion spin probes based on trinitrophenol, but increased the binding of positively charged hydrophobic ion probes based on triphenylalkylphosphoniums. They also enhanced the transit rates for both hydrophobic anions and cations; however, translocation rates were enhanced to a greater extent for the cation probes compared to the anion probes. The changes in binding constant for cations versus anions could be accurately accounted for using a simple model for the free energy profile for hydrophobic ions across membranes, and indicate that these anesthetics decrease the membrane dipole potential. From a fit of the experimental data to this model, anesthetics could promote a decrease in the dipole potential in two ways. First, anesthetics appear to modify the effective dipole moment in the membrane interface and may accomplish this by orienting their molecular dipole antiparallel to the intrinsic dipoles at the interface. Second, they modify the membrane dielectric constant, leading to a decrease in the field across the interface. At equivalent membrane concentrations, isoflurane, enflurane, and halothane produced similar changes in the dipole potential and decreased the dipole potential as much as 65 mV at a membrane mole fraction of 0.20.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Membrane orientation of the N-terminal segment of alamethicin determined by solid-state 15N NMR.

Alamethicin was synthesized with 15N incorporated into alanine at position 6 in the peptide sequence. In dispersions of hydrated dimyristoylphosphatidylcholine, solid-state 15N NMR yields an axially symmetric powder pattern indicating that the peptide is reorienting with a single axis of symmetry when associated with lamellar lipids. When incorporated into bilayers that are uniformly oriented with the bilayer normal parallel to the B(o) field, the position of the observed 15N chemical shift is 171 ppm. This is coincident with the sigma parallel to edge of the axially symmetric powder pattern for non-oriented hydrated samples. Thus the axis of motional averaging lies along the bilayer normal. Two-dimensional separated local field spectra were obtained that provide a measure of the N-H dipolar coupling in one dimension and the 15N chemical shift in the other. These data yield a dipolar coupling of 17 kHz corresponding to an average angle of 24 degrees for the N-H bond with respect to the B(o) field axis. An analysis of the possible structures and orientations that could produce the observed spectral parameters show that these values are consistent with an alpha-helical conformation inserted along the bilayer normal.

Alamethicin↗
PubMed1994

Structure of micelle-associated alamethicin from 1H NMR. Evidence for conformational heterogeneity in a voltage-gated peptide.

Alamethicin is a 20 amino acid peptide that produces a voltage-dependent conductance in membranes. To understand the mechanism by which this peptide becomes voltage-gated, the structure of alamethicin bound to micelles was examined using high-resolution 1H nuclear magnetic resonance (NMR). Two-dimensional correlation and nuclear Overhauser effect spectroscopy (NOESY) were carried out on alamethicin incorporated into perdeuterated sodium dodecyl sulfate (SDS) micelles, and the 1H NMR spectrum of the peptide in micelles was assigned. The intensities of the HN-HN(i,i+1), H alpha-HN(i,i+1), H alpha-NH(i,i+3), H alpha-H beta (i,i+3), and H alpha-NH(i,i+4) cross peaks in the NOESY spectrum suggest that the N-terminal half of the peptide is predominantly alpha-helical, while the C-terminal half has a less regular or more flexible structure. The exposure of micelle bound alamethicin to the aqueous solution was determined by examining the effect of aqueous paramagnetic reagents on the line widths of the peptide protons. These measurements suggest that alamethicin is buried in the micelle. A set of restraints consisting of 175 distances (derived from NOESY spectra), five dihedral angles, and two hydrogen bond distances were used in a simulated annealing procedure that yielded structures for micelle associated alamethicin. The structures that were generated with simulated annealing were largely helical from residues 4-9 and 12-16. A limited number of structural forms were obtained. The main difference among forms involved the backbone conformations of MeA10, Gly11, and Leu12 and resulted in structures that were straight or had different amounts of bend. The structural forms could be easily interconverted by rotation of the psi and phi angles of residues 10-12. The rotational freedom at or near MeA10 may be a result of Pro14, which would be the normal hydrogen-bonding position for the peptide carbonyl of MeA10. These results suggest that conformation rearrangements at or near MeA10 may play a role in the voltage-gating of alamethicin.

Alamethicin↗
PubMed1994

Collisions between helical peptides in membranes monitored using electron paramagnetic resonance: evidence that alamethicin is monomeric in the absence of a membrane potential.

Alamethicin is a 20-amino-acid peptide that produces a voltage-dependent conductance in membranes. We investigated the state of aggregation of alamethicin in egg phosphatidylcholine and dioleoylphosphatidylcholine membranes by examining the EPR spectra obtained from an active analog of this peptide that is spin-labeled at its C-terminus. The dependence of both the linewidth and signal intensity as a function of peptide concentration exhibit exchange broadening as the peptide concentration is increased; however, the exchange rates are linear with peptide concentration as is expected for the simple diffusion of monomers. In addition, the spin-exchange rates obtained from the linebroadening are consistent with collisional rates that are predicted from free Brownian diffusion. The results provide strong evidence that in the absence of a membrane potential, alamethicin is largely monomeric in these membranes.

Alamethicin↗
PubMed1994

Molecular flexibility demonstrated by paramagnetic enhancements of nuclear relaxation. Application to alamethicin: a voltage-gated peptide channel.

A nitroxide spin label attached to the C-terminus of the channel forming peptide alamethicin produces an enhancement of the nuclear spin-lattice relaxation rates of peptide protons as a result of both intermolecular and intramolecular magnetic dipole-dipole interactions. The intermolecular contribution provides evidence that alamethicin monomers collide preferentially in a C-terminal-to-N-terminal configuration in methanol. From the intramolecular paramagnetic enhancement of nuclear spin-lattice relaxation times, effective distances between the unpaired electron on the nitroxide at the C-terminus of alamethicin and protons along the peptide backbone were calculated. These distances are much shorter than distances based on the reported crystal structure of alamethicin, and cannot be accounted for by motion in the bonds that attach the nitroxide to the peptide. In addition, the differences between distances deduced from the nuclear spin relaxation and the distances seen in the crystal structure increase toward the N-terminal end of the peptide. The simplest explanation for these data is that the alamethicin backbone suffers large structural fluctuations that yield shorter effective distances between the C-terminus and positions along the backbone. This finding can be interpreted in terms of a molecular mechanism for the voltage-gating of the alamethicin channel. When the distances between a paramagnetic center and a nucleus fluctuate, paramagnetic enhancements are expected to yield distances that are weighted by r-6, and distances calculated using the Solomon-Bloembergen equations may more nearly represent a distance of closest approach than a time average distance. Therefore, the use of paramagnetic centers such as spin labels or metal ions with long electron T1 values provides a distance measurement that reflects a dynamically averaged structure where the averaging process heavily weights short distances. The results of such measurements, when combined with other structural information, may provide particularly clear evidence for the magnitude of structural fluctuations involving distances greater than 10 A.

Alamethicin↗
PubMed1993

Internal electrostatic potentials in bilayers: measuring and controlling dipole potentials in lipid vesicles.

The binding and translocation rates of hydrophobic cation and anion spin labels were measured in unilamellar vesicle systems formed from phosphatidylcholine. As a result of the membrane dipole potential, the binding and translocation rates for oppositely charged hydrophobic ions are dramatically different. These differences were analyzed using a simple electrostatic model and are consistent with the presence of a dipole potential of approximately 280 mV in phosphatidylcholine. Phloretin, a molecule that reduces the magnitude of the dipole potential, increases the translocation rate of hydrophobic cations, while decreasing the rate for anions. In addition, phloretin decreases the free energy of binding of the cation, while increasing the free energy of binding for the anion. The incorporation of 6-ketocholestanol also produces differential changes in the binding and translocation rates of hydrophobic ions, but in an opposite direction to those produced by phloretin. This is consistent with the view that 6-ketocholestanol increases the magnitude of the membrane dipole potential. A quantitative analysis of the binding and translocation rate changes produced by ketocholestanol and phloretin is well accounted for by a point dipole model that includes a dipole layer due to phloretin or 6-ketocholestanol in the membrane-solution interface. This approach allows dipole potentials to be estimated in membrane vesicle systems and permits predictable, quantitative changes in the magnitude of the internal electrostatic field in membranes. Using phloretin and 6-ketocholestanol, the dipole potential can be altered by over 200 mV in phosphatidylcholine vesicles.

Anions↗
PubMed1993

Probes of membrane electrostatics: synthesis and voltage-dependent partitioning of negative hydrophobic ion spin labels in lipid vesicles.

Two spin-labeled derivatives of the hydrophobic anion trinitrophenol have been synthesized and characterized in lipid vesicles. In the presence of lipid vesicles, the electron paramagnetic resonance (EPR) spectra of these probes are a composite of both membrane-bound and aqueous populations; as a result, the membrane-aqueous partitioning can be determined from their electron paramagnetic resonance spectra. The effect of transmembrane potentials on the membrane-aqueous partitioning of these spin-labeled hydrophobic ions was examined in phosphatidylcholine vesicles formed by extrusion. Inside positive membrane potentials promote an increase in the binding of these probes that is quantitatively accounted for by a simple thermodynamic model used previously to describe the partitioning of paramagnetic phosphonium ions. The transmembrane migration rates of these ions are dependent on the dipole potential, indicating that these ions transit the membrane in a charged form. The partitioning of the probe is also sensitive to the membrane surface potential, and this dependence is accurately accounted for using the Gouy-Chapman Stern formalism. As a result of the membrane dipole potential, these probes exhibit a stronger binding and a more rapid transmembrane migration rate compared with positive hydrophobic ion spin labels and provide a new set of negatively charged hydrophobic ion probes to investigate membrane electrostatics.

Biophysical Phenomena↗
PubMed1992

Determination of the molecular dynamics of alamethicin using 13C NMR: implications for the mechanism of gating of a voltage-dependent channel.

Alamethicin is a channel-forming peptide antibiotic that produces a highly voltage-dependent conductance in planar bilayers. To provide insight into the mechanisms for its voltage dependence, the dynamics of the peptide were examined in solution using nuclear magnetic resonance. Natural-abundance 13C spin-lattice relaxation rates and 13C-1H nuclear Overhauser effects of alamethicin were measured at two magnetic field strengths in methanol. This information was interpreted using a model-free approach to obtain values for the overall correlation times as well as the rates and amplitudes of the internal motions of the peptide. The picosecond, internal motions of alamethicin are highly restricted along the peptide backbone and indicate that it behaves as a rigid helical rod in solution. The side chain carbons exhibit increased segmental motion as their distance from the peptide backbone is increased; however, these motions are not unrestricted. Methyl group dynamics are also consistent with the restricted motions observed for the backbone carbons. There is no evidence from these dynamics measurements for a hinged motion of the peptide about proline-14. Alamethicin appears to be slightly less structured in methanol than in the membrane; as a result, alamethicin is also expected to behave as a rigid helix in the membrane. This suggests that the gating of this peptide involves changes in the orientation of the entire helix, rather than the movement of a segment of the peptide backbone.

Alamethicin↗
PubMed1992

Comparison of the lipid acyl chain dynamics between small and large unilamellar vesicles.

13C NMR spin-lattice relaxation (T1) rates and 13C-1H nuclear Overhauser effects (NOEs) were measured in an identical fashion in two lipid preparations having dramatically different curvatures. The T1 times that were obtained at four magnetic field strengths were fit along with the NOEs to simple models for lipid molecular dynamics. The results indicate that phospholipid chain ordering and dynamics are virtually identical in small and large unilamellar vesicles at the time scales sampled by these 13C-NMR studies. The order parameters and reorientational correlation times that characterize the amplitudes and rates of internal acyl chain motions were equal within experimental error for the methylene segments in the middle of the chains. The only significant differences in order parameters and correlation times between the two vesicle types were small and appeared at the ends of the acyl chains. At the carbonyl end the order was slightly higher in small vesicles than large vesicles, and at the methyl end the order was slightly lower for small vesicles. This indicates that in the more planar systems the acyl chains exhibit a slightly flatter order profile than in more highly curved membranes. The use of the same experimental approach in both small and large vesicle systems provided a more reliable and accurate assessment of the effect of curvature on molecular order than has been previously obtained.

Carbon Isotopes↗
PubMed1991

Voltage-dependent conductance for alamethicin in phospholipid vesicles. A test for the mechanism of gating.

The ion currents induced by alamethicin were investigated in unilamellar vesicles using electron paramagnetic resonance probe techniques. The peptide induced currents were examined as a function of the membrane bound peptide concentration, and as a function of the transmembrane electrical potential. Because of the favorable partitioning of alamethicin to membranes and the large membrane area to aqueous volume in vesicle suspensions, these measurements could be carried out under conditions where all the alamethicin was membrane bound. Over the concentration range examined, the peptide induced conductances increased approximately with the fourth power of the membrane bound peptide concentration, indicating a channel molecularity of four. When the alamethicin induced currents were examined as a function of voltage, they exhibited a superlinear behavior similar to that seen in planar bilayers. Evidence for the voltage-dependent conduction of alamethicin was also observed in the time dependence of vesicle depolarization. These observations indicate that the voltage-dependent behavior of alamethicin can occur in the absence of a voltage-dependent phase partitioning. That is, a voltage-dependent conformational rearrangement for membrane bound alamethicin leads to a voltage-dependent activity.

Alamethicin↗