New tools to advance biophysical study and inhibition of peripheral membrane proteins.

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Abstract Peripheral membrane proteins (PMPs) represent crucial mediators of biological and disease processes. This class of proteins exists in a water-soluble state until targeted to adhere on membranes, enabling them to perform their function. As with any membrane associated protein, PMPs are challenging to study, particularly in their membrane bound state, leaving many basic questions about function unresolved. Additionally, utilizing PMPs as drug targets is difficult since current methods are designed for water-soluble proteins and do not work well for membrane associated proteins. Thus, there is a great need for novel tools and procedures to illuminate details of PMP function and to create PMP inhibitors for use in chemical biology study and as drug leads. The goal of our research is to enable high-resolution, quantitative study of PMP interactions and allow inhibitor design for this elusive type of protein. We will initially focus our efforts on three PMPs of extraordinary biomedical interest: glutathione peroxidase 4 (GPx4) and Phox homology (PX) domains in the NADPH oxidase family (p47phox-PX and NOXO1-PX). We have initiated development of a novel membrane model, membrane- mimicking reverse micelles (mmRMs), which is based on the chemistry of cellular membranes. PMPs embed into mmRMs as they do with cellular membranes, enabling high-resolution study using NMR spectroscopy and other techniques. mmRMs have a number of advantages over current models, including greatly enhanced stability, outstanding spectroscopic properties, and an ability to house high concentrations of analyte along with the protein. We will modify our mmRM system to better reflect a variety of cellular and organelle membranes, allowing the system to be tuned according to the natural PMP environment. Our focus will then be to harness the unique properties of our mmRMs to enable unrivaled detail in study and quantification of PMP interactions with membranes and lipid substrates. To facilitate inhibitor design, we will develop a novel method that allows fragment screening of membrane-embedded PMPs, for which current methods are not suitable. Using these tools, we will initiate an inhibitor design campaign for our important PMP targets. Overall, our goal is to develop tools that will enable breakthroughs in detailed study of PMP biology as well as inhibitor and drug development for this largely untapped class of proteins.