Binding of imipramine to phospholipid bilayers using radioligand binding assay

Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined with centrifugation or filtration. Lipid bilayers were composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS). IMI binding isotherms were measured up to IMI concentration of 0.5 mmol/l. Due to electrostatic attraction, binding between the positively charged IMI and the negatively charged surfaces of PS membranes was augmented compared to binding to neutral PC membranes. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherms were described both by surface partition coefficients and by binding parameters (association constants and binding capacities). It was confirmed that binding of IMI to model membranes is strongly affected by negatively charged phospholipids and that the binding is heterogeneous; in fact, weak surface adsorption and incorporation of the drug into the hydrophobic core of lipid bilayer can be seen and characterized. These results support the hypothesis suggesting that the lipid part of biological membranes plays a role in the mechanism of antidepressant action.     

Model phospholipid membranes affect the holomyoglobin structure: conformational changes at pH 6.2

The influence of model negatively charged membranes on the sperm whale holomyoglobin structure at pH 6.2 has been investigated by different techniques (far and near UV circular dichroism, tryptophan fluorescence, absorbance at Soret band, differential scanning microcalorimetry and fast performance liquid chromatography). It is shown that the holomyoglobin structure undergoes a conformational transition from the native to intermediate state analogous to its apo-form. This state is characterized by the absence of a rigid tertiary structure and the native heme environment. At the same time, the content of alpha-helical secondary structure remains almost native. To change the holomyoglobin structure similarly to that of its apo-form in the presence of membranes, a higher molar phospholipids/protein ratio is required. The properties of holomyoglobin in the presence of negatively charged membranes resemble those of the molten globule state of its apo-form protein in aqueous solution. A possible functional role of the discovered non-native myoglobin state is discussed.     

Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states

The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists in equilibrium between folded and unfolded states under non-denaturing buffer conditions. In order to examine the origins of this instability, we have made mutations in the domain and characterized the thermodynamics and kinetics of folding. Results of substitutions of negatively charged residues to neutral amino acid residues suggest that the large electrostatic potential of the domain does not play a dominant role in the instability of the domain. Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwise highly conserved glycine residue in the diverging beta-turn connecting the beta3 and beta4 strands. Mutation of T22 to glycine results in significant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further characterize the basis for the stabilization of the T22 mutant relative to wild-type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native beta-turn structure at this position. Correlation of folding rates with AGADIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, suggests that the free energy of the unfolded state also plays a role in stability. This result highlights the importance of both folded and unfolded states for understanding protein stability.     

Lipid-induced conformational transition of the amyloid core fragment Abeta(28-35) and its A30G and A30I mutants

The interaction of the beta-amyloid peptide (Abeta) with neuronal membranes could play a key role in the pathogenesis of Alzheimer's disease. Recent studies have focused on the interactions of Abeta oligomers to explain the neuronal toxicity accompanying Alzheimer's disease. In our study, we have investigated the role of lipid interactions with soluble Abeta(28-35) (wild-type) and its mutants A30G and A30I in their aggregation and conformational preferences. CD and Trp fluorescence spectroscopic studies indicated that, immediately on dissolution, these peptides adopted a random coil structure. Upon addition of negatively charged 1,2-dipalmitoyl-syn-glycero-3-phospho-rac-(glycerol) sodium salt (PG) lipid, the wild-type and A30I mutant underwent reorganization into a predominant beta-sheet structure. However, no conformational changes were observed in the A30G mutant on interaction with PG. In contrast, the presence of zwitterionic 1,2-dipalmitoyl-syn-glycero-3-phosphatidylcholine (PC) lipid had no effect on the conformation of these three peptides. These observations were also confirmed with atomic force microscopy and the thioflavin-T assay. In the presence of PG vesicles, both the wild-type and A30I mutant formed fibrillar structures within 2 days of incubation in NaCl/P(i), but not in their absence. Again, no oligomerization was observed with PC vesicles. The Trp studies also revealed that both ends of the three peptides are not buried deep in the vesicle membrane. Furthermore, fluorescence spectroscopy using the environment-sensitive probe 1,6-diphenyl-1,3,5-hexatriene showed an increase in the membrane fluidity upon exposure of the vesicles to the peptides. The latter effect may result from the lipid head group interactions with the peptides. Fluorescence resonance energy transfer experiments revealed that these peptides undergo a random coil-to-sheet conversion in solution on aging and that this process is accelerated by negatively charged lipid vesicles. These results indicate that aggregation depends on hydrophobicity and propensity to form beta-sheets of the amyloid peptide, and thus offer new insights into the mechanism of amyloid neurodegenerative disease.     

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid binding of synthetic talin peptides provides evidence for an intrinsic membrane anchor of talin

Talin, an actin-binding protein, is assumed to anchor at the membrane via an intrinsic amino acid sequence. Three N-terminal talin fragments, 21-39 (S19), 287-304 (H18), and 385-406 (H17) have been proposed as potential membrane anchors. The interaction of the corresponding synthetic peptides with lipid model systems was investigated with CD spectroscopy, isothermal titration calorimetry, and monolayer expansion measurements. The membrane model systems were neutral or negatively charged small unilamellar vesicles or monolayers with a lateral packing density of bilayers (32 mN/m). S19 partitions into charged monolayers/bilayers with a penetration area A(p) = 140 +/- 30 A(2) and a free energy of binding of DeltaG(0) = -5.7 kcal/mol, thereby forming a partially alpha-helical structure. H18 does not interact with lipid monolayers or bilayers. H17 penetrates into neutral and charged monolayers/bilayers with A(p) = 148 +/- 23 A(2) and A(p) = 160 +/- 15 A(2), respectively, forming an alpha-helix in the membrane-bound state. Membrane partitioning is mainly entropy-driven. Under physiological conditions the free energy of binding to negatively charged membranes is DeltaG(0) = -9. 4 kcal/mol with a hydrophobic contribution of DeltaG(h) = -7.8 kcal/mol, comparable to that of post-translationally attached membrane anchors, and an electrostatic contribution of DeltaG(h) = -1.6 kcal/mol. The latter becomes more negative with decreasing pH. We show that H17 provides the binding energy required for a membrane anchor.     

Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P₂. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P₂ or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170 μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.     

Presence of anionic phospholipids rules the membrane localization of phenothiazine type multidrug resistance modulator

Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are mainly cationic amphiphiles. The molecular mechanism of their action can involve interactions with transporter proteins as well as with membrane lipids. The interactions between anionic phospholipids and MDR modulators can be crucial for their action. In present work we study interactions of 2-trifluoromethyl-10-(4-[methanesulfonylamid]buthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS). Using microcalorimetry, steady-state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug location in membrane depends on lipid type. The electrostatic attraction between drug and lipid headgroups presumably keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral ones. FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures. Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures resemble pure PC ones.     

The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.     

Understanding the Mechanism of Prosegment-catalyzed Folding by Solution NMR Spectroscopy

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.     

Rigidification of a flexible protease inhibitor variant upon binding to trypsin

The Tyr35-->Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 A resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 A data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.     

The Conformational State of Apomyoglobin in the Presence of Phospholipid Vesicles at Neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near and far UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Fusion of liposomes containing a novel cationic lipid, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium: induction by multivalent anions and asymmetric fusion with acidic phospholipid vesicles

The fusion behavior of large unilamellar liposomes composed of N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) and either phosphatidylcholine (PC) or phosphatidylethanolamine (PE) has been investigated by a fluorescence resonance energy transfer assay for lipid mixing, dynamic light scattering, and electron microscopy. Polyvalent anions induced the fusion of DOTMA/PE (1:1) liposomes with the following sequence of effectiveness: citrate greater than EDTA greater than phosphate, in the presence 100 mM NaCl, pH 7.4. Sulfate, dipicolinate, and acetate were ineffective. DOTMA/PC (1:1) vesicles were completely refractory to fusion in the presence of multivalent anions in the concentration range studied, consistent with the inhibitory effect of PC in divalent cation induced fusion of negatively charged vesicles. DOTMA/PE vesicles could fuse with DOTMA/PC vesicles in the presence of high concentrations of citrate, but not of phosphate. Mixing of DOTMA/PE liposomes with negatively charged phosphatidylserine (PS)/PE or PS/PC (1:1) vesicles resulted in membrane fusion in the absence of multivalent anions. DOTMA/PC liposomes also fused with PS/PE liposomes and, to a limited extent, with PS/PC liposomes. These observations suggest that the interaction of the negatively charged PS polar group with the positively charged trimethylammonium of DOTMA is sufficient to mediate fusion between the two membranes containing these lipids and that the nature of the zwitterionic phospholipid component of these vesicles is an additional determinant of membrane fusion.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

Model phospholipid membranes affect the tertiary structure of holomyoglobin: Conformational changes at pH 6.2

The influence of model negatively charged membranes on the structure of sperm whale holomyoglobin at pH 6.2 has been investigated using a variety of techniques (far-UV and near-UV circular dichroism, tryptophan fluorescence, absorption spectroscopy, differential scanning microcalorimetry, and fast-performance liquid chromatography). It has been shown that, similarly to apomyoglobin, holomyoglobin in the presence of phospholipid vesicles undergoes a conformational transition from the native to the intermediate state, which is characterized by loss of the rigid tertiary structure and the native heme environment; at the same time, the content of -helical secondary structures remains virtually unchanged. The molar phospholipid/protein ratio required for this transition is higher than in the case of apomyoglobin. The properties of holomyoglobin in the presence of negatively charged membranes are largely similar to those of the molten globule state of its apo form in aqueous solution. A possible functional role of this novel non-native state of myoglobin in the cell is discussed.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 120–128.Original Russian Text Copyright © 2005 by Basova, Tiktopulo, Bychkova.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics.

A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild-type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of the wild-type protein has a half-time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 "DTT-sensitive" mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the native wild-type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated at most sites without completely preventing folding. Because this genetic screen is based on changes in folding energetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.