Jean Marie Ruysschaert

Attenuated total reflection infrared spectroscopy of proteins and lipids in biological membranes

2. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 2.1. Preparation of ¢lms by evaporation of the solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 2.2. Immersion of ¢lms in bulk liquid environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 2.3. Preparation of ¢lm by Langmuir-Blodgett transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 2.4. Adsorption from bulk phase method on Langmuir-Blodgett ¢lms . . . . . . . . . . . . . . . . 113 2.5. Preparation of pure protein ¢lms in an aqueous environment by adsorption on the IRE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 2.6. Observation of a ¢lm in situ by external re£ection . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 2.7. Depth pro¢ling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

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).

Evidence of a specific complex between adriamycin and negatively-charged phospholipids

Membrane-model systems (monolayers, small unilamellar vesicles) were used to study the interaction between adriamycin (ADM) and phospholipids. Adsorption of 3H-labeled adriamycin on different phospholipid monolayers demonstrated the specificity of adriamycin for negatively-charged phospholipids (cardiolipin, phosphatidylserine, phosphatidic acid). The stoichiometry has been found to be approx. 2 mol (1.8) adriamycin per mol cardiolipin and approx. 1 mol (0.75) adriamycin per mol phosphatidylserine and phosphatidic acid. No adsorption was detected with neutral lipids. Surface-potential measurements confirm the formation of a complex stabilized by electrostatic interactions without penetration of the drug into the lipid lipophilic phase. Some adriamycin derivatives were used to discriminate between the ionized hydrophilic and hydrophobic contributions in the complex formation. The absorption spectrum of adriamycin in the presence of cardiolipin resembles the behavior of the ADM-DNA complex. Moreover, the association constants of the two complexes are very similar (cardiolipin-ADM, 1.6 . 10(6) . M-1; ADM-DNA, 2.4 . 10(6) . M-1). To explain the high affinity of cardiolipin for adriamycin, we proposed that two essential interactions are responsible for the complex stabilization: an electrostatic interaction between the protonated amino groups of the sugar residues and the ionized phosphate residues, and an interaction between adjacent anthraquinone chromophores. These data strongly suggest competitive behavior between a membrane site and the target. Consequently, it must be assumed that the lipidic components of the cell membrane structure may be an important determinant in the behavior of adriamycin. This observation should be kept in mind in the building of new derivatives.

Antiparallel beta-sheet: a signature structure of the oligomeric amyloid beta-peptide

AD (Alzheimers disease) is linked to Abeta (amyloid beta-peptide) misfolding. Studies demonstrate that the level of soluble Abeta oligomeric forms correlates better with the progression of the disease than the level of fibrillar forms. Conformation-dependent antibodies have been developed to detect either Abeta oligomers or fibrils, suggesting that structural differences between these forms of Abeta exist. Using conditions which yield well-defined Abeta-(1-42) oligomers or fibrils, we studied the secondary structure of these species by ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy. Whereas fibrillar Abeta was organized in a parallel beta-sheet conformation, oligomeric Abeta displayed distinct spectral features, which were attributed to an antiparallel beta-sheet structure. We also noted striking similarities between Abeta oligomers spectra and those of bacterial outer membrane porins. We discuss our results in terms of a possible organization of the antiparallel beta-sheets in Abeta oligomers, which may be related to reported effects of these highly toxic species in the amyloid pathogenesis associated with AD.

Structural Characterization of the Hydrophobin SC3, as a Monomer and after Self-Assembly at Hydrophobic/Hydrophilic Interfaces

Hydrophobins are small fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes that, in the case of Class I hydrophobins, can be disassembled only by treatment with agents like pure trifluoroacetic acid. Here we characterize, by spectroscopic techniques, the structural changes that occur upon assembly at an air/water interface and upon assembly on a hydrophobic solid surface, and the influence of deglycosylation on these events. We determined that the hydrophobin SC3 from Schizophyllum commune contains 16-22 O-linked mannose residues, probably attached to the N-terminal part of the peptide chain. Scanning force microscopy revealed that SC3 adsorbs specifically to a hydrophobic surface and cannot be removed by heating at 100 degrees C in 2% sodium dodecyl sulfate. Attenuated total reflection Fourier transform infrared spectroscopy and circular dichroism spectroscopy revealed that the monomeric, water-soluble form of the protein is rich in beta-sheet structure and that the amount of beta-sheet is increased after self-assembly on a water-air interface. Alpha-helix is induced specifically upon assembly of the protein on a hydrophobic solid. We propose a model for the formation of rodlets, which may be induced by dehydration and a conformational change of the glycosylated part of the protein, resulting in the formation of an amphipathic alpha-helix that forms an anchor for binding to a substrate. The assembly in the beta-sheet form seems to be involved in lowering of the surface tension, a potential function of hydrophobins.

The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids

We used a 32P‐labeled pCMV‐CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV‐CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3‐tetradecylamino‐N‐tert‐butyl‐N′‐tetradeccyl‐propionamidine, and previously described as diC14‐amidine [1]), [2] lipofectin (a 1:1 mixture of N‐(1‐2,3‐dioleyloxypropyl)N,N,N‐triethylammonium (DOTMA) and dioleylphosphatid‐ylethanolamine (DOPE)), and [3] DMRIE‐C (a 1:1 mixture of N‐[1‐(2,3‐dimyristyloxy)propyl]‐N,N‐dimethyl‐N‐(2‐hydroxyethyl) ammonium bromide (DMRIE) and cholesterol). Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE‐C. Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA‐cationic lipid complex entry into COS cells for the three cationic lipids. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE‐C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA‐lipofectin complexes. This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes.

Cationic liposomal lipids: from gene carriers to cell signaling

Cationic lipids are positively charged amphiphilic molecules which, for most of them, form positively charged liposomes, sometimes in combination with a neutral helper lipid. Such liposomes are mainly used as efficient DNA, RNA or protein carriers for gene therapy or immunization trials. Over the past decade, significant progress has been made in the understanding of the cellular pathways and mechanisms involved in lipoplex-mediated gene transfection but the interaction of cationic lipids with cell components and the consequences of such an interaction on cell physiology remains poorly described. The data reported in the present review provide evidence that cationic lipids are not just carriers for molecular delivery into cells but do modify cellular pathways and stimulate immune or anti-inflammatory responses. Considering the wide number of cationic lipids currently available and the variety of cellular components that could be involved, it is likely that only a few cationic lipid-dependent functions have been identified so far.

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.

Adriamycin inactivates cytochrome c oxidase by exclusion of the enzyme from its cardiolipin essential environment.

Abstract Adriamycin and several derivatives were found to inhibit the last oxidation site of the respiratory chain (cytochrome c oxidase EC.1.9.3.1.) both on mitochondria and on purified reconstituted systems. A new mechanism of membrane enzyme inactivation is proposed to explain the experimental results: adriamycin does not interact directly with cytochrome c oxidase but inactivates it by changing the cardiolipin environment essential for its activity. In presence of adriamycin, cardiolipin is extracted from the lipid surrounding environment of cytochrome c oxidase and segregates in a separate phase inaccessible for the enzyme. We suggest that other cardiolipin dependent enzymes could be inactivated by adriamycin.

Secondary and Tertiary Structure Changes of Reconstituted P-glycoprotein A FOURIER TRANSFORM ATTENUATED TOTAL REFLECTION INFRARED SPECTROSCOPY ANALYSIS

The structure of purified P-glycoprotein functionally reconstituted into liposomes was investigated by attenuated total reflection Fourier transform infrared spectroscopy. A quantitative evaluation of the secondary structure and a kinetic of 2H/H exchange of the P-glycoprotein were performed both in the presence and in the absence of MgATP, MgATP-verapamil, and MgADP. This approach was previously shown to be a useful tool to detect tertiary structure changes resulting from the interaction between a protein and its specific ligands, as established for the Neurospora crassa H+-ATPase. 2H/H exchange measurements provided evidence that a large fraction of the P-glycoprotein is poorly accessible to the aqueous medium. Addition of MgATP induced an increased accessibility to the solvent of a population of amino acids, while addition of MgATP-verapamil resulted in a subtraction of a part of the protein from access to the aqueous solvent. No significant changes were observed upon addition of MgADP or verapamil alone. The secondary structure of P-glycoprotein was not affected by addition of ligands. The variations observed in the 2H/H exchange rate when P-glycoprotein interacted with the above ligands therefore represented tertiary structure changes. Fluorescence quenching experiments confirmed that MgATP-induced changes are to be found in the tertiary structure of the enzyme.