Véronique Cabiaux

Determination of Soluble and Membrane Protein Structure by Fourier Transform Infrared Spectroscopy

The basic knowledge accumulated over the last twenty years on the different vibrations of polypeptides were reviewed in Chapter 8. Because of the complexity of naturally occurring proteins, most of these data have been obtained from the study of model compounds, from simple amino acid derivatives to large synthetic polypeptides, which can be crystallized in a single secondary structure. This chapter covers biologically synthesized proteins. Data on this subject are much more recent because the advent of the new generation of Fourier transform spectrophotometers only now provides high quality spectra. Simultaneously, manipulations of the spectra have been made possible by the concomitant digitalization of the spectra and the availability of low cost computers in laboratories. It was only in 1986 that the race for determination of secondary structure from manipulated IR spectra started with a paper by Byler and Susi (1986), although it is only fair to say that the results of several attempts to obtain secondary structures had been published before. The number of papers using infrared spectros-copy (IR) to obtain secondary structures has been growing exponentially ever since. One purpose of the present review is to point out, through the description and the comparison of the different methods, that interpretation of the results still needs caution. Indeed, while some spectral features of the main secondary structures are well established, others are not. Moreover, no agreement exists on a “correct” mathematical treatment of the spectra. Both the intrinsic uncertainties in the assignments and the methodological diversity open the door to flawed conclusions if the user is not properly aware of these problems. Such warnings have been issued previously (Haris and Chapman, 1992; Surewicz et al., 1993; Haris and Chapman, 1992).

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops

‘Type III secretion’ allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore‐forming properties of the lipid‐bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild‐type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild‐type bacteria. The observed channel could be responsible for the translocation of Yop effectors.

Translocation of Bacillus anthracis lethal and oedema factors across endosome membranes.

The two exotoxins of Bacillus anthracis, the causative agent of anthrax, are the oedema toxin (PA–EF) and the lethal toxin (PA–LF). They exert their catalytic activities within the cytosol. The internalization process requires receptor‐mediated endocytosis and passage through acidic vesicles. We investigated the translocation of EF and LF enzymatic moieties across the target cell membrane. By selective permeabilization of the plasma membrane with Clostridium perfringens delta‐toxin, we observed free full‐size lethal factor (LF) within the cytosol, resulting from specific translocation from early endosomes. In contrast, oedema factor (EF) remained associated with the membranes of vesicles.

Purification of IpaC, a protein involved in entry of Shigella flexneri into epithelial cells and characterization of its interaction with lipid membranes.

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the secreted IpaA–D proteins. A complex containing IpaC and IpaB is able to promote uptake of inert particles by epithelial cells. This suggested that Ipa proteins, either individually or as a complex, might interact with the cell membrane. We have purified IpaC and demonstrated its interaction with lipid vesicles. This interaction is modulated by the pH, which might be relevant to the dual role of Ipa proteins, in induction of membrane ruffles upon entry and lysis of the endosome membrane thereafter.

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.

Interaction with a lipid membrane: a key step in bacterial toxins virulence.

Bacterial toxins are secreted as soluble proteins. However, they have to interact with a cell lipid membrane either to permeabilize the cells (pore forming toxins) or to enter into the cytosol to express their enzymatic activity (translocation toxins). The aim of this review is to suggest that the strategies developed by toxins to insert in a lipid membrane is mediated by their structure. Two categories, which contains both pore forming and translocation toxins, are emerging: alpha helical proteins containing hydrophobic domains and beta sheets proteins in which no hydrophobicity can be clearly detected. The first category would rather interact with the membrane through multi-spanning helical domains whereas the second category would form a beta barrel in the membrane.

Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH

Like diphtheria toxin and the N‐terminal (M r 23 000) region of fragment B, CB1 (M r 13 000), the cyanogen bromide peptide located in the middle region of fragment B is able to induce pore formation in lipid bilayer membrane at low pH. These two peptides (M r 23 000 and 13 000) share a common segment (M r 6300) containing the predicted amphipathic, α‐helical, transverse lipid‐associating domain (M r 2750) of fragment B[J. Cell Biol. (1980) 87, 837–840]. Therefore, we postulated this domain to be responsible for the pore formation ability of diphtheria toxin [Proc. Natl. Acad. Sci. USA (1981) 78, 172–176]. A relationship between the pH dependency of pore formation and the presence of a cluster of prolines in the C‐terminal region of CB1 is proposed.

Diphtheria toxin induces fusion of small unilamellar vesicles at low pH

Model membrane systems such as phospholipid vesicles have been extensively used to study the mechanism of membrane fusion at the molecular level. We report here on the capacity of diphtheria toxin to induce fusion of small unilamellar vesicles of dipalmitoylphosphatidylcholine at low pH. Fluorescence polarization and differential scanning calorimetry make it possible to demonstrate the mixing of the lipid phase. Mixing of the internal aqueous compartments of liposome was established using the terbium fluorescence technique. The analogy of structure and properties between melittin and a diphtheria toxin fragment is discussed.

Topology of diphtheria toxin B fragment inserted in lipid vesicles

Diphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells In response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptides that were protected from digestion were separated by gel electrophoresls, transferred to a membrane and their N‐terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194–223, 266–375 and 429–528. The secondary structures of the peptides inserted in the membrane, determined by Fourier‐transformed infrared spectroscopy, were shown to be mostly α‐helices and β‐sheets (44% and 53%, respectively). On the basis of these data and the recently published X‐ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.

Orientation of gramicidin A at the lysophosphatidylcholine / water interface: a semi-empirical conformational analysis

Abstract The mode of orientation of gramicidin A in the β 6.3 conformation in a monolayer of lysophosphatidylcholine was studied on the basis of conformational data. The approach involves localization of the centers of hydrophobicity and hydrophilicity of the gramicidin molecule, and takes into account the dielectric constant discontinuity of the interface. The most probable orientation is that with the C-terminal, tryptophan-rich, end directed towards the hydrophobic medium. In this orientation, four lysophosphatidylcholines surround a gramicidin monomer leading to a complex with an overall cylindrical shape compatible with the bilayer structure found in aqueous dispersions of mixtures of these molecules (Killian, J.A., De Kruijff, B., Van Echteld, C.J.A., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1983) Biochim. Biophys. Acta 728, 141–144).