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Integrated Atomic Force Microscopy Techniques for Analysis of Biomaterials : : Study of Membrane Proteins

  • Author(s): Connelly, Laura S.
  • et al.
Abstract

Atomic Force Microscopy (AFM) is the prominent techniques for structural studies of biological materials in physiological relevant fluidic environments. AFM has been used to resolve the three-dimensional (3D) surface structure of cells, membranes, and proteins structures. Ion channels, formed by membrane proteins, are the key structures that control the activity of all living systems. This dissertation focuses on the structural evaluation of membrane proteins through atomic force microscopy. In Part I, AFM is utilized to study one of the most prominent medical issues facing our society, Alzheimer's Disease (AD). AD is a misfolded protein disease characterized by the accumulation of [Beta]- amyloid (A[Beta]) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic channel structures composed of A[Beta] peptides. Selectively engineered sequences of A[Beta] were examined by AFM to elucidate the substructures and thus activity A[Beta] channels. Key residues were evaluated with the intent better understand the exact nature by which these pores conduct electrical and molecular signals, which could aid in identifying potential therapeutic targets for the prevention/treatment of AD. Additionally, AFM was used to analyze brain derived A[Beta] and newly developed pharmacological agents to study membranes and A[Beta]. Part II, presents a novel technology that incorporates electrophysiology into the AFM interface, enabling simultaneous imaging and complementary conductance measurements. The activity of ion channels is studied by various techniques, including patch clamp, free standing lipid bilayers, droplet interface bilayers, and supported lipid bilayers. However, direct correlation with channel structures has remained a challenge. The integrated atomic force microscopy system presented offers a solution to this challenge. The functionality of the system is demonstrated with an Sf9 membrane plaque containing Cx26

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