Membrane-induced Conformational Changes Research Articles

Schistocins: Novel antimicrobial peptides encrypted in the Schistosoma mansoni Kunitz Inhibitor SmKI-1

BackgroundHere we describe a new class of cryptides (peptides encrypted within a larger protein) with antimicrobial properties, named schistocins, derived from SmKI-1, a key protein in Shistosoma mansoni survival. This is a multi-functional protein with biotechnological potential usage as a therapeutic molecule in inflammatory diseases and to control schistosomiasis. MethodsWe used our algorithm enCrypted, to perform an in silico proteolysis of SmKI-1 and a screening for potential antimicrobial activity. The selected peptides were chemically synthesized, tested in vitro and evaluated by both structural (CD, NMR) and biophysical (ITC) studies to access their structure-function relationship. ResultsEnCrypted was capable of predicting AMPs in SmKI-1. Our biophysical analyses described a membrane-induced conformational change from random coil-to-α-helix and a peptide-membrane equilibrium for all schistocins. Our structural data allowed us to suggest a well-known mode of peptide-membrane interaction in which electrostatic attraction between the cationic peptides and anionic membranes results in the bilayer disordering. Moreover, the NMR H/D exchange data with the higher entropic contribution observed for the peptide-membrane interaction showed that schistocins have different orientations upon the membrane. ConclusionsThis work demonstrate the robustness for using the physicochemical features of predicted peptides in the identification of new bioactive cryptides. Besides, it demonstrates the relevance of combining these analyses with biophysical methods to understand the peptide-membrane affinity and improve further algorithms. General significanceBioprospecting cryptides can be conducted through data mining of protein databases demonstrating the success of our strategy. The peptides-based agents derived from SmKI-1 might have high impact for system-biology and biotechnology.

Conformation-specific perturbation of membrane dynamics by structurally distinct oligomers of Alzheimer's amyloid-β peptide.

The accumulation of toxic soluble oligomers of the amyloid-β peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease. There are mainly two conformationally distinct oligomers, namely, prefibrillar and fibrillar oligomers, that are recognized by conformation-specific antibodies, anti-amyloid oligomer antibody (A11) and anti-amyloid fibrillar antibody (OC), respectively. Previous studies have shown that the interaction of Aβ oligomers with the lipid membrane is one of the key mechanisms of toxicity produced by Aβ oligomers. However, the mechanism by which structurally distinct Aβ oligomers interact with the lipid membrane remains elusive. In this work, we dissect the molecular mechanism underlying the interaction of structurally distinct Aβ42 oligomers with the lipid membrane derived from the brain total lipid extract. Using picosecond time-resolved fluorescence spectroscopy, we show that the A11-positive Aβ42 oligomers undergo a membrane-induced conformational change that promotes the deeper immersion of these oligomers into the lipid hydrocarbon region and results in an increase in the membrane micro-viscosity. In sharp contrast, OC-positive Aβ42 oligomers interact with the lipid membrane via electrostatic interactions between the negatively-charged lipid headgroup and positively-charged residues of Aβ42 without perturbing the membrane dynamics. We show that the two structurally distinct Aβ42 oligomers demonstrating different interaction mechanisms with the lipid membrane eventually lead to the formation of typical amyloid fibrils. Our findings provide the mechanistic underpinning of the perturbation of lipid membranes by two conformationally distinct Aβ42 oligomers and can be of prime importance in designing anti-Alzheimer's therapeutic agents targeting Aβ-membrane interactions.

Probing protein-induced membrane fouling with in-situ attenuated total reflectance fourier transform infrared spectroscopy and multivariate curve resolution-alternating least squares

Proteins are one of the major contributors to membrane fouling. The interaction between proteins and the polymer membrane at the molecular level is essential for the alleviation/prevention of membrane fouling, but remains unclear. In this work, time-dependent in-situ attenuated total reflectance Fourier transform infrared spectroscopy is applied to investigate the interaction process between two model proteins, bovine serum albumin and lysozyme, and the poly(vinylidene fluoride) (PVDF) membrane. Multivariate curve resolution-alternating least squares is integrated with two-dimensional correlation spectroscopy analysis to resolve the membrane-induced conformational changes of proteins. The multivariate curve resolution-alternating least squares analysis reveals a two-step process in the protein-membrane interaction and provides the kinetics of the conformational transition, which aids the segmentation of the spectral dataset. By applying two-dimensional correlation spectroscopy analysis to different groups of the time-dependent spectra, the sequential order of the secondary structural changes of proteins is determined. The proteins initially undergo unfolding transition to a more open, less structured state, which appears to be triggered by the hydrophobic membrane surface. Afterwards, the proteins become aggregated with the high anti-parallel β-sheet content, aggravating the membrane fouling. The conformational transition process of proteins was also confirmed by the atomic force microscopic images and quartz crystal microbalance measurement. Overall, this work provides an in-depth understanding of the interaction between proteins and the membrane surface, which is helpful for the development of membrane anti-fouling strategies.

Red blood cell membrane-facilitated release of nitrite-derived nitric oxide bioactivity.

The reduction of nitrite by deoxyhemoglobin to nitric oxide (NO) has been proposed as a mechanism for the transfer of NO bioactivity from the red blood cell (RBC) to the vasculature. This transfer can increase vascular dilatation. The major challenge to this hypothesis is the very efficient scavenging of NO by hemoglobin, which prevents the release of NO from RBCs. Previous studies indicate that the reaction of nitrite with deoxyhemoglobin produces two metastable intermediates involving nitrite bound to deoxyhemoglobin and a hybrid intermediate [Hb(II)NO(+) ↔ Hb(III)NO] where the nitrite is reduced, but unavailable to react with hemoglobin. We have now shown how unique properties of these intermediates provide a pathway for the release of NO bioactivity from RBCs. The high membrane affinity of these intermediates (>100-fold greater than that of deoxyhemoglobin) places these intermediates on the membrane. Furthermore, membrane-induced conformational changes of the nitrite-reacted intermediates facilitate the release of NO from the hybrid intermediate and nitrite from the nitrite-bound intermediate. Increased membrane affinity, coupled with facilitated dissociation of NO and nitrite from the membrane-bound intermediates, provides the first realistic mechanism for the potential release of NO and nitrite from the RBC and their potential transfer to the vasculature.

Small-angle neutron scattering reveals the assembly of alpha-synuclein in lipid membranes

The aggregation of α-synuclein (asyn), an intrinsically disordered protein (IDP), is a hallmark in Parkinson's disease (PD). We investigated the conformational changes that asyn undergoes in the presence of membrane and membrane mimetics using small-angle neutron scattering (SANS). In solution, asyn is monomeric and unfolded assuming an ensemble of conformers spanning extended and compact conformations. Using the contrast variation technique and SANS, the protein scattering signal in the membrane–protein complexes is selectively highlighted in order to monitor its conformational changes in this environment. We showed that in the presence of phospholipid membranes asyn transitions from a monodisperse state to aggregated structures with sizes ranging from 200 to 900Å coexisting with the monomeric species. Detailed SANS data analysis revealed that asyn aggregates have a hierarchical organization in which clusters of smaller asyn aggregates assemble to form the larger structures. This study provides new insight into the mechanism of asyn aggregation. We propose an aggregation mechanism in which stable asyn aggregates seed the aggregation process and hence the hierarchical assembly of structures. Our findings demonstrate that membrane-induced conformational changes in asyn lead to its heterogeneous aggregation which could be physiologically relevant in its function or in the diseased state.

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin: MOLECULAR INSIGHTS INTO A CRITICAL PREREQUISITE OF MEMBRANE-BOUND MONOMERS

The insecticidal feature of the three-domain Cry δ-endotoxins from Bacillus thuringiensis is generally attributed to their capability to form oligomeric pores, causing lysis of target larval midgut cells. However, the molecular description of their oligomerization process has not been clearly defined. Here a stable prepore of the 65-kDa trypsin-activated Cry4Ba mosquito-specific toxin was established through membrane-mimetic environments by forming an ∼200-kDa octyl-β-D-glucoside micelle-induced trimer. The SDS-resistant trimer caused cytolysis to Sf9 insect cells expressing Aedes-mALP (a Cry4Ba receptor) and was more effective than a toxin monomer in membrane perturbation of calcein-loaded liposomes. A three-dimensional model of toxin trimer obtained by negative-stain EM in combination with single-particle reconstruction at ∼5 nm resolution showed a propeller-shaped structure with 3-fold symmetry. Fitting the three-dimensional reconstructed EM map with a 100-ns molecular dynamics-simulated Cry4Ba structure interacting with an octyl-β-D-glucoside micelle showed relative positioning of individual domains in the context of the trimeric complex with a major protrusion from the pore-forming domain. Moreover, high-speed atomic force microscopy imaging at nanometer resolution and a subsecond frame rate demonstrated conformational transitions from a propeller-like to a globularly shaped trimer upon lipid membrane interactions, implying prepore-to-pore conversion. Real-time trimeric arrangement of monomers associated with L-α-dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid bicelle membranes was also envisaged by successive high-speed atomic force microscopy imaging, depicting interactions among three individual subunits toward trimer formation. Together, our data provide the first pivotal insights into the structural requirement of membrane-induced conformational changes of Cry4Ba toxin monomers for the molecular assembly of a prepore trimer capable of inserting into target membranes to generate a lytic pore.

Molecular Details of α-Synuclein Membrane Association Revealed by Neutron Reflectometry

Understanding how environmental factors affect α-synuclein conformation is of great importance because its misfolding is intimately connected to Parkinson's disease etiology. The role of membranes is of particular interest because membranes not only affect the protein-folding landscape, but are also ubiquitous in vivo. We have employed neutron reflectometry techniques to characterize membrane-induced conformational changes in α-synuclein, revealing the polypeptide structural envelope and changes in bilayer structure. In our previous study (1), the partial insertion of α-synuclein induced membrane thinning of a negatively charged tethered bilayer composed of equal molar phosphatidylcholine (PC) and phosphatidic acid (PA). We have extended this work to study a physiologically more abundant anionic lipid, phosphatidylserine (PS) and used isotopically labeled deuterated protein to enhance signal contrast between the protein and the bilayer. In order to enhance α-synuclein binding to PS, measurements were conducted at an acidic pH, 5.5, which yielded a similar binding affinity to that of PA at pH 7 as determined by circular dichroism spectroscopy detecting α-helical structure formation. Similar to PA, neutron data obtained from tethered PC/PS bilayer also showed α-synuclein induced membrane thinning in a protein concentration dependent manner. Towards the aim to delineate the involvement of specific protein regions, we have produced a segmental isotopically-labeled α-syn variant where the N-terminal (residues 1-86) and C-terminal regions (residues S87C-140) are deuterated and protonated, respectively. Having the N-terminal region deuterated, the contrast between the protonated lipid and the peptide is easily distinguishable. Results obtained from complementary fluorescence methods to determine penetration depth and lipid reorganization will also be presented.1. Pfefferkorn, C.M., Heinrich, F., Sodt, A.J., Maltsev, A.S., Pastor, R.W., Lee, J.C. (2012) Depth of α-synuclein in a bilayer determined by fluorescence, neutron reflectometry, and computation, Biophys. J. 102, 613-621.

Capturing Spontaneous Membrane Insertion and Membrane-Induced Conformational Changes of Talin at an Atomic Resolution

Integrins are a diverse set of proteins that play a central role in complex biological processes, such as tumor metastasis and thrombus formation. The integrin heterodimer is often expressed in a low-affinity, inactive state, relying on specific cytoplasmic or extracellular signals for its activation. The cytoskeletal-associated protein talin constitutes one of the major activation pathways of integrin through a membrane-mediated mechanism. While the involvement of activated, membrane-bound talin in this process is well established, atomic details of membrane binding of talin and of talin-dependent integrin activation have been lacking. Using our novel, highly mobile membrane mimetic simulation system, we have successfully captured complete insertion of the talin head domain (THD) in a phosphatidylserine membrane in three independent unbiased simulations, revealing key molecular events involved in the process. The THD is initially recruited to the membrane via the documented membrane orientation patch (MOP), consisting of a large number of positively charged residues. Electrostatic potential calculations revealed THD to be highly polarized, providing a potential mechanism explaining how the protein is aligns for optimal encounter with the membrane. We also observe a large, membrane-induced interdomain conformational change (>2.5 nm), which brings the F3 subdomain into contact with the anionic membrane via residues K325, N326, and K327. This result explains how F2 and F3 subdomains can simultaneously bind the membrane, a biochemically established aspect that could not be explained by the crystal structure. Moreover, we characterize a phenylalanine-rich region as the hydrophobic membrane anchor, consisting mainly of F261 and F283, which is released through the snorkeling motion of a few critical lysine residues within the membrane. Although such an anchor has been hypothesized to exist, none had been identified prior to this study.

The Cytosolic Domain of Fis1 Binds and Reversibly Clusters Lipid Vesicles

Every lipid membrane fission event involves the association of two apposing bilayers, mediated by proteins that can promote membrane curvature, fusion and fission. We tested the hypothesis that Fis1, a tail-anchored protein involved in mitochondrial and peroxisomal fission, promotes changes in membrane structure. We found that the cytosolic domain of Fis1 alone binds lipid vesicles, which is enhanced upon protonation and increasing concentrations of anionic phospholipids. Fluorescence and circular dichroism data indicate that the cytosolic domain undergoes a membrane-induced conformational change that buries two tryptophan side chains upon membrane binding. Light scattering and electron microscopy data show that membrane binding promotes lipid vesicle clustering. Remarkably, this vesicle clustering is reversible and vesicles largely retain their original shape and size. This raises the possibility that the Fis1 cytosolic domain might act in membrane fission by promoting a reversible membrane association, a necessary step in membrane fission.

Induced Conformational Changes in the Activation of the Pseudomonas aeruginosa type III Toxin, ExoU

ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca 2+-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.

Membrane Insertion and Membrane-Induced Conformational Changes of Talin F2F3 Triggering Integrin Activation

Activation of the integrin αβ heterodimer plays an important role in important biological processes such as thrombus formation and tumor metastasis. A well-known activator of intergin is the cyoskeletal-associated protein talin, whose membrane association is suggested to initiate the activation process. However, atomic details of the membrane-binding event and subsequent activation of integrin are largely unknown. We have utilized a novel membrane mimetics system developed in our laboratory, in conjunction with equilibrium molecular dynamics (MD) simulations to describe the membrane-binding event of talin F2F3 and characterize its subsequent interaction with integrin β3. In addition to characterizing the membrane orientation patch of talin F2F3, we were able to elucidate a conserved, Phe-rich hydrophobic anchor which was suggested by mutational studies, but was not evident in previous structural studies. Additionally, crystallographic studies could not explain the binding of the F3 subdomain to the membrane, which is well-characterized in mutational studies. Utilizing the highly-mobile membrane mimetics, we were able to capture large-scale conformational changes in talin F2F3 which promote the interaction of the F3 subdomain with the membrane via several residues in the β1-β2 loop. This study represents one of the first examples of membrane-induced large-scale conformational changes observed using MD simulations. Using the membrane-bound talin F2F3 subdomain, we then studied how the integrin β3 will be affected by membrane binding of talin. These simulations revealed atomistic details of the NPxY-PTB binding motif as well as formation of the membrane-proximal hydrophobic pocket. Additionally, talin F2F3 induces the bending of integrin β3, changing the angle between the cytoplasmic and transmembrane domains by approximately 50°. This buries the conserved β-Asp within the headgroup region, possibly giving a mechanism as to how the α-β salt bridge is broken and consequently how integrin is activated.

Interaction of prothrombin with a phospholipid surface: evidence for a membrane-induced conformational change

Prothrombin interacts with phosphatidylserine containing platelet membranes via its N-terminal, γ-carboxyglutamate (gla) residue-rich domain. Once bound it is cleaved to form the active protease, thrombin (factor IIa). Human prothrombin was cleaved with cathepsin G in the absence of calcium and magnesium ions. Under these conditions, the gla domain was removed. Phospholipid protected the protein from this proteolytic event, and this suggests that a conformational change may be induced by interaction with phospholipids. Binding of prothrombin to a surface containing 20% phosphatidylserine/80% phosphatidylcholine was detected by surface plasmon resonance, whereas no interaction with gla-domainless prothrombin was observed. Binding of intact prothrombin in the presence of calcium ions showed complex association kinetics, suggesting multiple modes of initial interaction with the surface. The kinetics of the dissociation phase could be fitted to a two-phase, exponential decay. This implies that there are at least two forms of the protein on the surface one of which dissociates tenfold more slowly than the other. Taken together, these data suggest that, on binding to a membrane surface, prothrombin undergoes a conformational change to a form which binds more tightly to the membrane.

Membrane-Induced Conformational Changes of Kyotorphin Revealed by Molecular Dynamics Simulations

The analgesic dipeptide kyotorphin (l-Tyr-l-Arg) was studied in the two most relevant protonation states at physiological pH, both in water and in a membrane model, using molecular dynamics simulations. Kyotorphin is found to exhibit a remarkable conformational freedom even when strongly interacting with the bilayer. Nevertheless, we observe a strong decrease in the population of the tyrosine's chi(1) torsion angle around 60 degrees that could be correlated with the dipeptide biological function. We employed a linear response approximation methodology to determine the N-terminus pK(a) values of kyotorphin and obtained 7.80 and 7.94 for aqueous and lipidic systems, respectively. Our results also indicate that the interaction of kyotorphin with a biological membrane model is consistent with the "membrane catalyst" hypothesis, and that even after the reduction of conformational freedom due to membrane insertion, this peptide fulfils most of the known constraints present in the opioid-like receptors.

Structural Determination and Tryptophan Fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a Fungal Glycolipid Transfer Protein (GLTP), Provide Novel Insights into Glycolipid Specificity and Membrane Interaction by the GLTP Fold

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 A) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp(66), Asn(70), Lys(73), Trp(109), and His(147)) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at approximately 355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (approximately 6 nm) permitting determination of binding affinities. The unique positioning of Trp(208) at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.

Characterization of Indolicidin-Membrane Interactions By Simultaneous Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy-Atomic Force Microscopy

A detailed understanding of how antimicrobial peptides interact with bacterial membranes is a key step towards the effective design of novel antibiotics to treat infection. These interactions may include membrane-induced conformational changes to the peptide, membrane disordering, as well as peptide aggregation. To understand the effect of both membrane composition and peptide sequence on these phenomena, we applied simultaneous attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR)-atomic force (AFM) microscopy to directly visualize and characterize the interactions of the model antimicrobial peptide, indolicidin, with a series of supported planar lipid bilayers. This approach allows us to directly interrogate how peptide association, aggregation, and insertion alter the structure of the bilayer. It also allows us to directly assess changes to the secondary structure of the peptide as a consequence of both specific peptide-membrane interactions as well as peptide-peptide interactions. Simultaneously acquired AFM images provide direct confirmation of the effect of the peptide on membrane integrity, evidence of domain targeting, as well as the kinetics and structure of putative peptide aggregates. This coupled approach provides a unique opportunity to directly link spectroscopic details associated with peptide-membrane interactions with structural insights obtained on nanometer length scales.

Membrane-induced peptide structural changes monitored by infrared and circular dichroism spectroscopy

As more peptide secondary structures deduced by infrared spectroscopy (IR) have been reported in the literature, there have been overlaps in assignments of elements of secondary structure to carbonyl vibrational frequencies. We have investigated this phenomenon with regards to the use of IR for monitoring membrane-induced structural changes using conformationally diverse peptides. These IR studies, complemented by circular dichroism (CD) experiments, revealed that peptide–solvent interactions can mask membrane-induced conformational changes monitored by IR. A structural transition from random coil to α-helix upon the binding of mastoparan X to a membrane was clearly observed by CD but obscured in the amide I region of the IR spectrum. In addition, unlike the buried helical peptides gramicidin D and P16 in micelles, the amide II peak for mastoparan X was absent, likely due to H–D exchange. This suggests information on the peptide's membrane-bound solvent accessibility could be obtained from this region of the spectrum.

Membrane-Induced Conformational Change of α1-Acid Glycoprotein Characterized by Vacuum-Ultraviolet Circular Dichroism Spectroscopy

The tertiary structure of alpha1-acid glycoprotein (AGP) remains unresolved despite its novel function because AGP is a hard target in X-ray and NMR analyses. To elucidate the membrane-induced conformational change of AGP, the vacuum-ultraviolet circular dichroism (VUVCD) spectra of AGP and its constituent sugars were measured down to 160 nm in the presence or absence of phosphoglyceride liposome using a synchrotron-radiation VUVCD spectrophotometer. The secondary-structure contents and numbers of segments of AGP were estimated from the VUVCD spectra of the protein moiety obtained by subtracting the contributions of the glycan moiety. Further, the positions of secondary structures on the amino acid sequence were predicted by combining the VUVCD data with a neural network algorithm. These comprehensive secondary-structure analyses revealed that AGP consists of 11.4% alpha-helices (3 segments) and 39.9% beta-strands (12 segments) in the absence of liposome (pH 4.5), which are close to the proportions in the secondary structure of native AGP (pH 7.4) predicted by homology modeling, and that it consists of 47.5% alpha-helices (7 segments) and 2.7% beta-strands (2 segments) in the presence of liposome (pH 4.5). Detailed characterization of these alpha-helices of AGP bound to liposome suggested that two alpha-helices (residues 15-27 and 161-175) in the N- and C-terminal regions strongly interact with liposome. Most of the progesterone-binding residues of AGP were involved in the sequences transferring from beta-strands to alpha-helices or unordered structures, which coincided with the large decrease in progesterone-binding capacity of liposome-bound AGP. These results provide the first sequence-level information on the membrane-binding mechanism and structure-function relationship of AGP.

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b 5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b 5 vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b 5 upon the interaction with its redox partners is discussed.

β-Sheet Structured β-Amyloid(1-40) Perturbs Phosphatidylcholine Model Membranes

The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-β peptides Aβ40 and Aβ42. These amphipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Aβ. Primarily unstructured Aβ has been shown to undergo a membrane-induced conformational change to either primarily β-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Aβ renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Aβ peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Aβ. Here, we have attempted incorporation of Aβ in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Aβ peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Aβ40 and Aβ42 are almost entirely helical, the Aβ analogs were β-structured in the resulting vesicle dispersions. When Aβ40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular “single channel-like” conductance of Aβ was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Aβ40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Aβ40 was present primarily as β-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small amphipathic Aβ40 assemblies.

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA(2)s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA(2)s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA(2), we have created semisynthetic human group IB PLA(2) in which the N-terminal decapeptide is joined with the (13)C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA(2) joined with a (13)C-labeled fragment of group IB PLA(2). Infrared spectral resolution of the unlabeled and (13)C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA(2) has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA(2) is more rigid than that of the IB PLA(2), but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA(2), which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA(2). Correlation is delineated between structural and membrane binding properties of PLA(2)s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA(2)s acts as a regulatory domain that mediates interfacial activation of these enzymes.