Solid State NMR Investigations of Lipid Bilayers and Biomembrane Binding Molecules: Dendrimers and Amylin.

Abstract

The goal of this project was to investigate the interaction between lipid binding molecules, such as peptides and drug compounds, with biological lipid bilayers. The results demonstrate that human amylin, a peptide hormone implicated in type II adult onset diabetes, disrupts cell membranes via a curvature inducing mechanism. In order to assess the effects that the various membrane-associated molecules have on lipid bilayers, a combination of differential scanning calorimetry (DSC) and solid state NMR methods were used. The amyloidogenic and toxic hIAPP1-37 peptide, the non-toxic and non-amyloidogenic rIAPP1-37 peptide, and the toxic but largely non-amyloidogenic rIAPP1-19 and hIAPP1-19 fragments were characterized. It is also shown that hyperbranched polymers with nanotherapeutic applications, known as poly(amidoamine) dendrimers, are thermodynamically stable when inserted inside zwitterionic lipid bilayers using 1H radio frequency driven dipolar recoupling (RFDR) and 1H magic angle spinning (MAS) nuclear Overhauser effect spectroscopy (NOESY) techniques. 14N and 31P NMR experiments on static samples and measurements of the mobility of C-H bonds using a 2D proton detected local field protocol under MAS corroborate these results. The localization of dendrimers in the hydrophobic core of lipid bilayers restricts the motion of bilayer lipid tails, with the smaller G5 dendrimer having more of an effect than the larger G7 dendrimer. Furthermore, solid state NMR techniques are developed to study the lipid bilayers and the molecules that associate with them. These methods drastically increase the spectral resolution of 2D solid state NMR techniques and allow more accurate structure and dynamics information to be extracted from solid phase NMR samples. These techniques, known as separated local field (SLF) techniques, are used to obtain information about the orientation dependent local fields at each chemical site in a molecule under investigation. SLF techniques have been very important in the extraction of structural information from aligned samples in the solid state. Furthermore, a method is proposed to enable resonance assignment to be made on uniformly labeled samples. This method promises to overcome many of the difficulties inherent in solid state NMR studies of the structure and dynamics of biological membranes and the molecules that associate with them.