Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH-as observed by liposome content leakage and circular dichroism experiments-and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.

Polyethylene glycol (PEG), a high-molecularweight colloid present in new organ preservation solutions, protects against cold ischemia injuries leading to better graft function of transplanted organs. This protective effect cannot be totally explained by immuno-camouflaging property or signaling-pathway modifications. Therefore, we sought for an alternative mechanism dependent on membrane fluidity. Using the Langmuir-Pockles technique, we show here that PEGs interacted with lipid monolayers of defined composition or constituted by a renal cell lipid extract. High-molecular-weight PEGs stabilized the lipid monolayer at low surface pressure. Paradoxically, at high surface pressure, PEGs destabilized the monolayers. Hypothermia reduced the destabilization of saturated monolayer whereas unsaturated monolayer remained unaffected. Modification of ionic strength and pH induced a stronger stabilizing effect of PEG 35,000 Da which could explain its reported higher effectiveness on cold-induced injuries during organ transplantation. This study sheds a new light on PEG protective effects during organ preservation different from all classical hypotheses.

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We have studied the interaction of trifluoperazine (TFP) with monolayers of various glycerophospholipids at 37 degrees C. TFP (1-10 microM) had little effect on surface pressure/molecular area isotherms in monolayers (on pure water) of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine but greatly increased the mean molecular area (mma) of dipalmitoylphosphatidylserine; the increment was greatest between 0 and 1 microM, and a further increase to 10 microM TFP gave only a slight increase in mma. With phosphatidylserine (PS)-containing stearoyl and varying acyls in the sn-1 and -2 positions, respectively, TFP increased the mma in a manner that depended on the number of double bonds and chain length. In mixtures of DPPC with two of these PS species the TFP-induced mma of the monolayers (on buffer, pH 7.4) increased linearly with the proportion of PS. Both PS and TFP have ionizable groups, and the TFP-induced mma increase had optima at pH 5.0 and 7.0. We conclude that the TFP-PS interaction is mainly, but not entirely, driven by electrostatic interactions between the TFP cation and PS headgroup anion, with an insertion of the phenothiazine moiety among the acyls in the monolayer that depends on the packing of the acyls.

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BackgroundThe regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B.Methodology/Principal FindingsAs revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212–216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers.Conclusions/SignificanceThermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation.

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