Abstract

Abstract

The effect of tricyclic antidepressants (TCA) on phospholipid bilayer structure and dynamics was studied to provide insight into the mechanism of TCA-induced intracellular accumulation of lipids (known as lipidosis). Specifically we asked if the lipid–TCA interaction was TCA or lipid specific and if such physical interactions could contribute to lipidosis. These interactions were probed in multilamellar vesicles and mechanically oriented bilayers of mixed phosphatidylcholine–phosphatidylglycerol (PC–PG) phospholipids using ³¹P and ¹⁴N solid-state NMR techniques. Changes in bilayer architecture in the presence of TCAs were observed to be dependent on the TCA's effective charge and steric constraints. The results further show that desipramine and imipramine evoke distinguishable changes on the membrane surface, particularly on the headgroup order, conformation and dynamics of phospholipids. Desipramine increases the disorder of the choline site at the phosphatidylcholine headgroup while leaving the conformation and dynamics of the phosphate region largely unchanged. Incorporation of imipramine changes both lipid headgroup conformation and dynamics. Our results suggest that a correlation between TCA-induced changes in bilayer architecture and the ability of these compounds to induce lipidosis is, however, not straightforward as imipramine was shown to induce more dramatic changes in bilayer conformation and dynamics than desipramine. The use of ¹⁴N as a probe was instrumental in arriving at the presented conclusions. Copyright © 2004 John Wiley & Sons, Ltd.

Human islet amyloid polypeptide (hIAPP), or amylin, is a 37 amino acid hormone secreted by pancreatic β-cells. hIAPP constitutes ∼90% of the amyloid deposits found in type II diabetic patients. It has been shown that the central region of the peptide (hIAPP_20–29) constitutes the nucleation site for the amyloidogenic process with F23 playing a key role in the formation of the β-pleated structures. In addition, it has been proposed that an important stage in the cytotoxicity of hIAPP is its interaction with the β-cell membranes. As a first step toward the characterization of the interaction of hIAPP with cell membranes, we determined conformational preferences of hIAPP_20–29 in membrane-mimicking environments. We found that upon interacting with negatively charged micelles, the dominant conformation of hIAPP_20–29 is a distorted type I β-turn centered on residues F23 and G24, with F23, A25, and I26 forming a small hydrophobic cluster that may facilitate the interaction of this peptide with the membrane bilayer. Moreover, we were able to elucidate the topological orientation of the peptide that is absorbed on the micelle surface, with the hydrophobic cluster oriented toward the hydrocarbon region of the micelles and both N- and C-termini exposed to the solvent. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 29–41, 2003

The relative stability of α-helix and β-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. ¹³C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the α-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% β-sheet and 45% α-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% β-sheet and 40% α-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% α-helix/50% β-sheet at 100°C when starting from a mostly α-helical state. No change was observed upon heating a β-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with β-sheet approximately 260 J/mol more stable than α-helix in solid-state PLA. © 2002 Wiley Periodicals, Inc. Biopolymers 64: 246–254, 2002