Structural Biology of Transmembrane Proteins

Project Collaborators:

Dr. Michael Malkowski Hauptman Woodward Institute, Dr. Michael Wiener University of Virginia

Flow chart for production, crystallization and structure determination of membrane
proteins by the Membrane Protein Structural Biology Consortium (MPSBC).

Despite their importance, approaches for analyzing the structures of transmembrane proteins are still slow and difficult compared to the standard technologies available for soluble proteins. Only about twenty structures of eukaryotic transmembrane proteins have been determined to date, compared with tens of thousands of structures of soluble proteins. We are attempting to determine high resolution x-ray structures of a variety of eukaryotic transmembrane proteins, as well as developing methods to facilitate membrane protein structure determination. This is being pursued as a component of the Membrane Protein Structural Biology Consortium (MPSBC), one of nine centers focused on membrane proteins funded as part of the Protein Structure Initiative of the National Institute of General Biomedical Sciences. The project focuses on structure determination of membrane transporters and membrane proteins involved in lipid metabolism and lipid modifications of proteins. The approaches being used include high-throughput cloning of orthologs of target proteins into yeast expression systems, extensive characterization of the properties of protein-detergent complexes, and new approaches for high-throughput crystallization trials.

Crystals of a transmembrane transporter.

An additional aspect of our investigation of the structural biology of transmembrane protein is the development of procedures for enhancing the thermodynamic stabilities of membrane proteins to facilitate the growth of crystals useful for structure determination. Many existing structures of membrane proteins have been solved using various mutational modifications of the target proteins that lock them in stable configurations. We have developed a genetic screen to identify cels expressing stabilized forms of receptors that retain high affinity for ligand at elevated temperatures. Stabilized receptors are being subjected to purification and crystallization for structure determination. We also expect that characterization of stabilizing mutations will provide better understanding of the poorly understand factors that govern the stability of the folded structures of membrane proteins.

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