José-Luis R. Arrondo

Infrared spectroscopy of phosphatidylcholines in aqueous suspension a study of the phosphate group vibrations

The 1000-1300 cm-1 region of the infrared spectrum of dipalmitoylphosphatidylcholine (DPPC) and other phosphate-containing molecules has been studied by the Fourier-transform technique. Three absorption bands have been assigned to various vibrational modes of the DPPC phosphate group, with maximum wavenumbers at 1060, 1086 and 1222 cm-1. These values are the same above and below Tc of the phospholipid. Dehydration produces band-shifts toward higher wavenumbers .

An infrared spectroscopic study of β-galactosidase structure in aqueous solutions

Fourier‐transform infrared spectroscopy has been used to elucidate the secondary structure of E. coli β‐galactosidase in aqueous solution. The structure of this enzyme was previously unknown above the level of the amino acid sequence. Spectra have been recorded in both H2O and D2O media; mutually complementing data are obtained, that provide unambiguous structural information. The results show that β‐galactosidase contains 40% β‐sheet and 35% α‐helical structure, with smaller proportions of random coil (12%) and β‐turns (13%).

Membrane-surfactant interactions The effect of triton X-100 on sarcoplasmic reticulum vesicles

The effect of Triton X-100 on purified sarcoplasmic reticulum vesicles has been studied by means of chemical, ultrastructural and enzymic techniques. At low detergent/membrane ratios (about 1 Triton X-100 per 60 phospholipid molecules) the only effect observed is an increase in vesicle permeability. Higher surfactant concentrations, up to a 1:1 detergent/phospholipid ratio, produce a large enhancement of ATPase activity. Membrane solubilization occurs as a critical phenomenon when the surfactant/phospholipid molar ratio reaches a value around 1.5:1, corresponding to 2 mumol Triton X-100/mg protein. At this point, the suspension turbidity drops, virtually all the protein and phospholipid is solubilized and every organized structure disappears. Simultaneously, a dramatic increase in the specific activity of the solubilized ATPase is observed. The sudden solubilization of almost all the bilayer components at a given detergent concentration is attributed to the relative simplicity of this membrane system. Solubilization takes place at the same surfactant/membrane ratio, at least between 0.5 and 4 mg membrane protein/ml. The non-solubilized residue seems to consist mainly of delipidized aggregated forms of ATPase.

Infrared evidence of a β-hairpin peptide structure in solution

The IR spectrum of an 16‐amino acid peptide corresponding, according to NMR studies, to a β‐hairpin has been analysed. Two characteristic features distinguish its spectrum from that of an antiparallel β‐sheet: the low‐frequency band that in a β‐sheet structure is located at 2 ∼ 1632 cm−1 appears here at 2∼ 1620 cm−1, and the high‐frequency component does not undergo the isotopic shift typical of β‐sheet from 1690 to 1675 cm−1 when transferred to D2O. The infrared characteristics associated with β‐hairpins have been described so far in two proteins, in one of which, whose three‐dimensional structure is known from X‐ray diffraction, a β‐hairpin has actually been detected.

Binding of β-amyloid (1-42) peptide to negatively charged phospholipid membranes in the liquid-ordered state: modeling and experimental studies.

To explore the initial stages of amyloid β peptide (Aβ42) deposition on membranes, we have studied the interaction of Aβ42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L(o)) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L(o) state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5-5 mol%) than higher (10-20 mol%) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aβ42 binding than L(o) ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of β-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aβ42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol% anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aβ42 and membrane.

Protein structural effects of agonist binding to the nicotinic acetylcholine receptor

The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier‐transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide 1 infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist‐induced alterations by band‐fitting analysis of the amide 1 spectral band reveals no appreciable changes in the percent of α‐helix, but a decrease in β‐sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration‐dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinerpic agonist implies significant rearrangements in the protein structure.

Interdomain Ca2+ effects in Escherichia coli α-haemolysin: Ca2+ binding to the C-terminal domain stabilizes both C- and N-terminal domains

alpha-Haemolysin (HlyA) is a toxin secreted by pathogenic Escherichia coli, whose lytic activity requires submillimolar Ca(2+) concentrations. Previous studies have shown that Ca(2+) binds within the Asp and Gly rich C-terminal nonapeptide repeat domain (NRD) in HlyA. The presence of the NRD puts HlyA in the RTX (Repeats in Toxin) family of proteins. We tested the stability of the whole protein, the amphipathic helix domain and the NRD, in both the presence and absence of Ca(2+) using native HlyA, a truncated form of HlyADeltaN601 representing the C-terminal domain, and a novel mutant HlyA W914A whose intrinsic fluorescence indicates changes in the N-terminal domain. Fluorescence and infrared spectroscopy, tryptic digestion, and urea denaturation techniques concur in showing that calcium binding to the repeat domain of alpha-haemolysin stabilizes and compacts both the NRD and the N-terminal domains of HlyA. The stabilization of the N-terminus through Ca(2+) binding to the C-terminus reveals long-range inter-domain structural effects. Considering that RTX proteins consist, in general, of a Ca(2+)-binding NRD and separate function-specific domains, the long-range stabilizing effects of Ca(2+) in HlyA may well be common to other members of this family.

Lipid-protein interactions. The mitochondrial complex III-phosphatidylcholine-water system

Bovine heart mitochondrial complex III (ubiquinol-cytochrome-c reductase) has been reconstituted into phosphatidylcholine bilayers and the effect of varying lipid/protein ratios on the structure and function of the protein has been examined. Electron microscopy, differential scanning calorimetry and Arrhenius plots of enzyme activity provide evidence that the protein is incorporated in an active conformation into pure phosphatidylcholine bilayers. At low lipid/protein ratios (e.g. 80:1 molar ratio) the protein exists in the form of aggregates. As the lipid proportion is increased, electron microscopy reveals the gradual formation of lipid bilayers; structures with the appearance of closed vesicles are seen at or above 300:1 phospholipid/protein molar ratios. Changes in enzyme activity as a function of lipid contents reveal a progressive increase in activity as more lipid is added, with a tendency to reach a saturation point. From the experimental data, a kinetic model is proposed, according to which the protein has an indefinite number of unspecific, independent and identical binding sites for phospholipids, the latter acting as essential enzyme activators. Varying lipid/protein ratios induce structural changes in complex III; visible spectra indicate changes in the polarity of the heme group environment, while Fourier-transform infrared spectroscopy suggests a change in the secondary structure of the protein as the lipid proportion is increased.

Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate A Fourier-transform infrared spectroscopic study

In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2+‐ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.

Interaction of the Nicotinic Acetylcholine Receptor with Ligands and Membrane Lipids Studied by Fourier-Transform Infrared Spectroscopy and Photoaffinity Labeling

Monitoring of the amide I band by Fourier-transform infrared spectroscopy (FT-IR) is a valid and flexible approach to monitor changes in the secondary structure of reconstituted Acetylcholine Receptor (AcChR). The continuous exposure of the AcChR to a cholinergic agonist (carbamylcholine), which drives the AcChR into the desensitized state, produces only minor changes in AcChR secondary structure. Nevertheless, carbamylcholine alters the AcChR tertiary or quaternary structure, as indicated by the increased thermal stability of the protein assessed from the temperature-dependence of the infrared spectrum. On the contrary, presence of a competitive cholinergic antagonist (d-tubocurarine) produces no detectable effects on AcChR structure. Cholesterol or the neut-al lipids present in asolectin extracts produce an ordering of the AcChR secondary structure observed by FT-IR and also enable the protein to increase its thermal stability in response to carbamylcholine. These effects of cholesterol or asolectin neutral lipids seem mediated by a direct interaction of all the AcChR subunits with the lipids, as suggested by labeling of the AcChR by a photoactivatable cholesterol analogue. The interaction between the AcChR and the cholesterol analogue is sensitive to AcChR desensitization since the presence of carbamylcholine during photolysis decreases the extent of labeling and alters the labeling stoichiometry in the AcChR subunits. This suggests the occurrence of an agonist-induced change in the arrangement of the transmembrane portion of the desensitized protein, which is consistent with the agonist-induced alteration of the AcChR protein tertiary or quaternary structure detected by FT-IR.