Membrane proteins are crucial to a wide variety of physiological processes, functioning as chemical receptors, transport channels and signal transducers. It is estimated that over half of drug targets are membrane proteins. Structural knowledge of membrane proteins is of very high biomedical significance. Crystallization of membrane proteins is an important approach to obtaining structural information of membrane proteins. However, obtaining diffracting crystals of membrane proteins is still difficult due to a number of bottlenecks, including low quantity and poor stability of membrane proteins;search of larger parameter space;and difficulty to obtaining diffraction sufficient to determine structures. No technology is currently available to address all of the needs and bottlenecks. SlipChip is an attractive and innovative technology that can potentially achieve these goals. It has been already applied to complex protocols such as enzyme assays, immunoassays, and PCR assays. The PI has demonstrated that SlipChip fabricated in glass can be used to handle and crystallize membrane proteins, and obtain crystals and high-resolution structures of soluble proteins. Yet, many technologies applicable to soluble proteins fail with membrane proteins;in addition, many elegant solutions to a single step in the crystallization workflow (e.g. establishing free interface diffusion) create additional bottlenecks elsewhere (e.g. creating problems in scale-up or crystal extraction). The goal of this Phase I proposal is to establish the feasibility and applicability of this innovation in the area of membrane proteins, while testing the feasibility of upstream and downstream steps in the crystallization workflow: from Aim 1) simple loading that will meter out nL volumes robustly for a range of solutions without the need for any equipment, to Aim 2) performance, from filling to incubation and observation, of chips made in plastic by inexpensive molding techniques, to Aim 3) robust methods of extraction and diffraction of crystals. Reaching these specific aims would firmly establish feasibility and viability of SlipChip technology for membrane protein crystallization, and would reduce the technical risk of Phase II work in this area, which would integrate many of the steps of concentration, separation, detergent exchange, purification, and formation of meso-phases into a single chip that accepts a fraction off a purification column and performs complex manipulations to produce diffraction-quality crystals of membrane proteins.
Public Health Relevance: Structural knowledge of membrane proteins is of very high biomedical significance to public health. Crystallization of membrane proteins is an important approach to obtaining their structural information, but is difficult due to a number of bottlenecks. This proposal describes a SlipChip technology to address unmet needs and bottlenecks for membrane protein crystallization.
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