The purpose of the project is to provide a new means for the production of integral membrane proteins that are good candidates for structure determination by X-ray crystallography. Structural information is essential to understand how enzymes and proteins function. Unfortunately, those proteins that are embedded in membranes are much more difficult to obtain in pure form and to crystallize. Although membrane proteins may comprise as much as 30% of the total number of proteins in a cell, the structures of membrane proteins make up less than 1% of depositions in the Protein Data Bank. Membrane proteins are particularly important to human health issues, since it is through these proteins that information as well as substances pass across various cellular membranes. While membrane proteins of mammalian origins have proven to be difficult to produce at quantities necessary for structural studies, homologous proteins from other species, including microorganisms, are more amenable to overproduction and purification. This has long been a successful strategy for basic research. Our project will focus on obtaining membrane proteins from thermophilic microorganisms that are adapted to life above 70oC. Proteins from these organisms tend to be quite stable at room temperature, and the probability of forming good quality crystals is increased in comparison to proteins from organisms that exist at lower temperatures. In the past few years, other groups have learned to manipulate the genetics of these organisms for the production of soluble proteins. Utilizing our expertise in membrane protein biochemistry, we aim to adapt these genetic tools for these extreme hyperthermophiles to overproduce their own membrane proteins with affinity tags attached. This method will greatly facilitate the purification of sample to enable crystallization efforts. We have selected three different extreme thermophiles that we will use to "manufacture" membrane proteins. We will pick at least 50 different proteins that are of particular interest, and produce them in affinity-tagged form within the thermophile. These proteins will be screened for crystallization conditions, and those that appear the most promising will be provided to the Center for Structures of Membrane Proteins to follow-up with the goal of complete structure determination. There are only about 218 structures of membrane proteins currently listed. We hope we can demonstrate a path towards significantly increasing this number.
The large majority of drug targets are proteins that reside in the membrane that surrounds our cells or organelles within our cells. Unfortunately, these proteins are the most difficult to obtain, limiting the pursuit of structural data. Many of these human proteins have counterparts in microorganisms that are adapted to life at very high temperatures, and these counterparts are particularly stable, easier to obtain, and to crystallize. We propose a new way to obtain these thermally stable proteins for the purposes of determining their structures, thus advancing health-related biomedical research.
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