Mechanism Of Membrane Insertion Research Articles

Probing interaction of melittin with lipid membranes using liquid secondary ion mass spectrometry

The membrane insertion mechanism of toxic protein is a very active domain in the study of molecular biology. and the “anchor state” of the membrane-hund protein on membrane is the key problem which it is difficult to solve with traditional methotls. In the present work we first studied the “anchor state” of melittin on membrane using liquid secondary ion mass spectrometry (LSIMS) in combination with proteolysis by specific enzyme. The results show that the membrane-bound melittin molecules mainly take the conformation in which the axis of α-helix lies parallel to the membrane surface and the side containing Lys7, Lys2I and Arg22 faces the outside of the lipid membrane. This discovery is very significant to the studies of membrane insertion mechanism. The results also indicate that the combination of mass spectrometry technique with the proteolysis by specific enzyme has provided a very new and effective method for the studies of the membrane insertion mechanism.

Calcium-dependent conformation of E. coli α-haemolysin. Implications for the mechanism of membrane insertion and lysis

Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli α-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca 2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca 2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95°C when bound to calcium, while it undergoes irreversible denaturation above 60°C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn 2+–HlyA interaction with the lipid bilayer has a strong polar component, while Ca 2+–HlyA appears to interact mainly through hydrophobic forces. Experiments in which HlyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca 2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca 2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.

Structure of a Cholesterol-Binding, Thiol-Activated Cytolysin and a Model of Its Membrane Form

The mechanisms by which proteins gain entry into membranes is a fundamental problem in biology. Here, we present the first crystal structure of a thiol-activated cytolysin, perfringolysin O, a member of a large family of toxins that kill eukaryotic cells by punching holes in their membranes. The molecule adopts an unusually elongated shape rich in β sheet. We have used electron microscopy data to construct a detailed model of the membrane channel form of the toxin. The structures reveal a novel mechanism for membrane insertion. Surprisingly, the toxin receptor, cholesterol, appears to play multiple roles: targeting, promotion of oligomerization, triggering a membrane insertion competent form, and stabilizing the membrane pore.

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy.

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a 15N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an alpha-helix content of approximately 42%. The alpha-helix comprises an uninterrupted hydrophobic stretch of < or = 12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15-75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled 15N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of 15N solid-state NMR spectroscopy (10(-4) seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed.

Folding intermediates of a beta-barrel membrane protein. Kinetic evidence for a multi-step membrane insertion mechanism.

The mechanism of folding and membrane insertion of integral membrane proteins, including helix bundle and beta-barrel proteins is not well understood. A key question is whether folding and insertion are coupled or separable processes. We have used the beta-barrel outer membrane protein A (OmpA) of Escherichia coli as a model to study the kinetics of folding and insertion into dioleoylphosphatidylcholine (DOPC) bilayers, as a function of temperature by gel electrophoresis, protease digestion, and fluorescence spectroscopy. OmpA was unfolded in 8 M urea solution (without detergent), and refolding and membrane insertion was initiated by rapid dilution of the urea concentration in the presence of phospholipid vesicles. In addition to the kinetically unresolved hydrophobic collapse in water, the time course of refolding of OmpA into DOPC bilayers exhibited three kinetic phases over a large temperature range. The first step was fast (k1 = 0.16 min-1) and not very dependent on temperature. The second step was up to two orders of magnitude slower at low temperatures (2 degrees C), but approached the rate of the first step at higher temperatures (40 degrees C). The activation energy for this process was 46 +/- 4 kJ/mol. A third slow process (k3 = 0.9 x 10(-2) min-1 at 40 degrees C) was observed at the higher temperatures. These results suggest that at least two membrane-bound intermediates exist when OmpA folds and inserts into lipid bilayers. We also show that both membrane-bound intermediates can be stabilized in fluid lipid bilayers at low temperatures. These intermediates share many properties with the adsorbed/partially inserted form of OmpA that was previously characterized in gel phase lipid bilayers [Rodionova et al. (1995) Biochemistry 34, 1921-1929]. Temperature jump experiments demonstrate, that the low-temperature intermediates can be rapidly converted to fully inserted native OmpA. On the basis of these and previous results, we present a simple folding model for beta-barrel membrane proteins, in which folding and membrane insertion are coupled processes which involve at least four kinetically distinguishable steps.

Major coat proteins of bacteriophage Pf3 and M13 as model systems for Sec-independent protein transport

The membrane insertion of bacteriophage coat proteins occurs independent of the Sec-translocase of Escherichia coli. Detailed study of the Pf3 and M13 coat proteins has elucidated two fundamental mechanisms of how proteins invade the membrane, most likely by direct interaction with the lipid bilayer. The Sec-independent translocation of amino-terminal regions across the inner membrane is limited to a short length and a small number of charged residues. Protein regions that contain several charged residues are efficiently translocated across the membrane when these regions are flanked by two adjacent hydrophobic segments interacting synergistically. The relevance of these findings for the membrane insertion mechanism of multispanning membrane proteins is discussed.

Major coat proteins of bacteriophage Pf3 and M13 as model systems for Sec-independent protein transport

The membrane insertion of bacteriophage coat proteins occurs independent of the Sec-translocase of Escherichia coli. Detailed study of the Pf3 and M13 coat proteins has elucidated two fundamental mechanisms of how proteins invade the membrane, most likely by direct interaction with the lipid bilayer. The Sec-independent translocation of amino-terminal regions across the inner membrane is limited to a short length and a small number of charged residues. Protein regions that contain several charged residues are efficiently translocated across the membrane when these regions are flanked by two adjacent hydrophobic segments interacting synergistically. The relevance of these findings for the membrane insertion mechanism of multispanning membrane proteins is discussed.

The domain structure of the ion channel-forming protein colicin Ia

Colicin Ia undergoes a transition from a soluble to a transmembrane state, forming an ion channel to effect its bactericidal activity. The X-ray crystal structure of soluble colicin Ia at an effective resolution of 4 A reveals that the molecule is highly alpha-helical and has an unusually elongated 'Y'-shape. The stalk and two arms of the 'Y' form three discrete structural domains which most likely correspond to the three functional regions identified for the channel-forming colicins. The channel-forming region of colicin Ia can be located to the larger of the two arms, the insertion domain, by its structural similarity to the ten alpha-helix motif found for the ion channel-forming fragments of colicins A and E1. The domain arrangement found in this structure provides novel insights into the mechanism of membrane insertion of colicin Ia.

Spontaneous insertion of polypeptide chains into membranes: a Monte Carlo model.

The Monte Carlo dynamics method was used to examine the process of protein insertion into model cell membranes. The water and lipid environments were taken into account via an effective medium approximation based on coordinate-dependent hydrophobic and hydrogen bond potentials. The polypeptide chain was represented in a full-backbone atom representation as a chain of diamond lattice vectors. The simulations support the idea that to a good approximation insertion may be depicted as a spontaneous thermodynamic process. The mechanism of membrane insertion of a simple lattice protein chain exhibits many features of theoretical predictions and is in good accord with experimental data. In the model, insertion begins with adsorption of the chain onto the interface, followed by the formation of helical fragments. These fragments, having partially saturated internal hydrogen bonds, can be transported into the lipid phase and then form transbilayer structures.

Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion.

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.

Insights into membrane insertion based on studies of colicins

The recently determined three-dimensional structure of the pore-forming domain of colicin A has led to a hypothetical model for membrane insertion and channel formation. Certain features of this model have implications for understanding the mechanism of membrane insertion by other toxins and may have a broader relevance to protein transport in general.

Alkaline phosphatase isozymes: recent progress

The past few years have witnessed the reports of significant new events in alkaline phosphatase (AP) isozymes. The cloning of the relevant genes and their nucleotide sequencing have all been accomplished. As a group, the genes for the intestinal, germ cell and placental isozymes have considerable sequence similarity; it is noteworthy that they occupy vicinal positions on chromosome 2, while the tissue unspecific AP gene is located on chromosome 1. The latter makes evolutionary lineage and instances of coordinate expression understandable. Another new development is the demonstration of a phosphatidyl inositol glycan tail on the C-terminus of these chromosome-2 AP genes. This is the major membrane insertion mechanism for AP, which is a cell surface membrane enzyme. This information may be helpful in understanding the phenomenon of the depletion of intestinal mucosal AP during fat absorption. Finally, a discussion has been focussed on recent studies on seminoma and AP, including immunodetection and immunoradiotherapy.

Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition.

The pH and temperature stabilities of diphtheria toxin and its fragments have been studied by high-sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37 degrees C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2 degrees C whereas that of the nucleotide-free toxin is centered at 51.8 degrees C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5 degrees C) and the B fragment (Tm = 58.4 degrees C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2 degrees C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane.

Inhibition of cytolysin activity in large granular lymphocyte granules by lipids: Evidence for a membrane insertion mechanism of lysis

The ability of various compounds to inhibit the lytic activity of purified cytoplasmic granules from LGL tumors was tested. The lytic activity of granule cytolysin was modestly inhibited by inorganic phosphate, and various monophosphoesters, with I 50 values in the range of 8–20 m M. Among monophosphoesters, choline phosphate was exceptionally potent, with an I 50 of 1.4m M. In contrast to the inhibition by phosphate esters, the parent compounds such as neutral sugars, glycerol, and choline, showed no detectable inhibition at 50 m M. A lysolipid bearing a short aliphatic chain and some detergents were inhibitory with I 50 values in the range of 1 m M. Lysolipids with longer aliphatic chains, phospholipids as liposomes, and related lipid compounds were found to display potent inhibition of the hemolytic activity of LGL granule cytolysin, with I 50 values in the range of 0.2–30 μ M. Soluble globular proteins inhibited LGL granule cytolysin hemolytic activity with I 50 of 0.07–0.4 mg/ml. However, lipoproteins were 2–3 orders of magnitude more potent inhibitors, with L 50 values of less than 1 μg/ml. The observed potent inhibition by lipid compounds lends support to a model of the lytic action of granule cytolysin in which soluble cytolysin molecules insert into the membrane lipid bilayer during the course of the lytic event.

An internal signal sequence: The asialoglycoprotein receptor membrane anchor

The human asialoglycoprotein receptor H1 is anchored in the membrane by a single stretch of 20 hydrophobic amino acids; the hydrophilic amino terminus faces the cytoplasm, and the carboxyl terminus is exoplasmic. We show here that glycosylation and insertion of the asialoglycoprotein receptor into the endoplasmic reticulum membrane is cotranslational and SRP-dependent and occurs without proteolytic cleavage. The membrane-anchor domain is necessary for membrane insertion, since a receptor with the segment deleted is neither inserted nor glycosylated. The segment is also sufficient for membrane insertion, since it will initiate translocation of a carboxy-terminal domain of rat α-tubulin across the membrane. We propose that a helical hairpin mechanism of membrane insertion is used both by cleaved amino-terminal and uncleaved internal signal sequences.

Hormone-induced loss of surface membrane during maturation of starfish oocytes: Differential effects on potassium and calcium channels

Prior to fertilization, starfish oocytes undergo meiotic maturation, triggered by the hormone 1-methyladenine (1-MA). Maturation involves a variety of complex biochemical, morphological, and electrical changes, many of which are similar to those caused by progesterone in vertebrates. Using voltage-clamp and ultrastructural techniques to study maturation in starfish, we have discovered a novel process by which 1-MA alters the electrical properties of the oocyte. The surface area of the oocyte decreases by more than 50% during the first hour of maturation, due to the elimination of microvilli, but the calcium and potassium currents present are affected differently by the loss of membrane. The amplitudes of both the transient K current (“A-current”) and the inwardly rectifying K current decrease, following the time course of the decrease in surface area, while the Ca current amplitude remains virtually unaffected, and may even increase in some oocytes. The kinetics of the currents do not change. This selective removal of K channels results in a larger and more rapidly rising action potential in the mature egg, which may aid in the fast block to polyspermy. The differential accessibility of various ion channels to mechanisms of membrane removal and insertion may play an important role in the development of excitable cells.

Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: relevance to the mechanism of action of colicins and certain toxins.

The channel-forming activity of colicin E1 in artificial membranes is known to increase at low pH values and to have a maximum near pH 4 in such membrane vesicles. The present work demonstrates that this pH dependence of activity can be attributed to membrane binding. Maximal binding of colicin E1 and a more slowly binding channel-forming carboxyl-terminal tryptic peptide occurred at acidic pH values, with the effective pK values for binding equal to 4.6 and less than 4.0, respectively. The binding did not require imposition of a transmembrane potential. Insertion of the tryptic peptide into the membrane was shown by retention of bound [3H]leucine-labeled peptide by vesicles after digestion with protease, as well as by retention of the peptide in salt-washed vesicles. The retention after protease treatment was also used to estimate the amount of carboxyl-terminal peptide inserted into the membrane. Approximately 12 of the 21 leucines present in the carboxyl-terminal peptide were retained after Pronase treatment at pH less than 4. Reversibility of the insertion at low pH values was seen after an alkaline shift of pH to 6.0, resulting in a decrease of the protease-inaccessible fraction of the bound protein. A model is presented describing a mechanism in which protonation of one or more carboxyl residues is necessary for effective binding and insertion into the membrane by the channel-forming domain of colicin E1. This model may also be relevant to the mechanism of membrane insertion by certain toxins.

Biophysical studies of ion channels.

Ionic channels, the integral membrane proteins responsible for the brain's electrical activity, have long been studied with standard electrophysiological and biochemical methods. Recently, however, newly developed electrical and molecular biological methods have been brought to bear on long-standing questions in neurobiology. Goals of current channel research include elucidating the primary amino acid sequence and three-dimensional structure of channel species; the mechanisms of synthesis, sorting, membrane insertion, and degradation; and aspects of function such as gating, ion permeation and selectivity, and regulation. The latest research combines the new biochemical and electrophysiological techniques to reveal relations between molecular structure and function.

Biosynthesis and transport of plasma membrane glycoproteins in the rat intestinal epithelial cell: studies with sucrase-isomaltase.

Sucrase-isomaltase (SI), an integral heterodimeric glycoprotein of the intestinal microvillus membrane, is synthesized as a single enzymically active precursor protein (pro-SI) of high relative molecular mass. After glycosylation in the Golgi complex pro-SI is transferred to the microvillus membrane where it is cleaved into the two subunits by pancreatic elastase. Pro-SI was purified by monoclonal antibody-affinity chromatography from microvillus membranes of fetal intestinal transplants in which SI is found exclusively in the non-cleaved precursor form. The N-terminal amino acid sequence of pro-SI was identical to that of the isomaltase subunit of SI which anchors the mature enzyme complex to the lipid bilayer, but it differed from the N-terminal sequence of the sucrase subunit of SI. This structural comparison indirectly gave insight into the mechanisms of membrane insertion and assembly of pro-SI during its biosynthesis. Subcellular fractionation studies indicate transient structural association of newly synthesized pro-SI with the basolateral membrane on its transfer from the Golgi complex to the microvillus membrane, suggesting that part of the basolateral membrane or its associated structures might be involved in the sorting-out processes of microvillar membrane proteins. This concept may have general relevance for the mechanisms of membrane insertion, intracellular transport and sorting of other microvillar membrane glycoproteins in the intestinal epithelial cell.

Thermodynamics, the structure of integral membrane proteins, and transport.

Membranes are structures whose lipid and protein components are at, or close to, equilibrium in the plane of the membrane, but are not at equilibrium across the membrane. The thermodynamic tendency of ionic and highly polar molecules to be in contact with water rather than with nonpolar media (hydrophilic interactions) is important in determining these equilibrium and nonequilibrium states. In this paper, we speculate about the structures and orientations of integral proteins in a membrane, and about how the equilibrium and nonequilibrium features of such structures and orientations might be influenced by the special mechanisms of biosynthesis, processing, and membrane insertion of these proteins. The relevance of these speculations to the mechanisms of the translocation event in membrane transport is discussed, and specific protein models of transport that have been proposed are analyzed.