PSI:Biology Profile Archive


MPSbyNMR: Paving the Way for High-Throughput Membrane Protein Structure Determination

Membrane Protein Structures by Solution NMR (MPSbyNMR), a PSI:Biology membrane protein structure determination center, is an Chou international collaboration of investigators from 6 organizations in 3 states and Germany. Under the direction of James Chou, Professor in Biological Chemistry & Molecular Pharmacology at Harvard Medical School, this center aims to develop an efficient solution NMR pipeline for solving membrane protein (MP) structures. Ten MP targets of unknown structure, including membrane-embedded transporters, enzymes, and receptors have been chosen to drive technology development and test the pipeline. All targets are polytopic helical MPs with 3-7 transmembrane helices and range in size from 18 – 43 kDa. “Using the proteins in this pipeline,” Chou said, “my colleagues and I plan to develop technologies that will have an immediate and practical impact on structure determination.”

Although the phrase “high-throughput” does not yet apply to membrane proteins in any technological context, MPSbyNMR aims to establish an NMR toolkit that has the capacity for systematic production of MP structures. Novel methods and improvements to existing protocols aimed at increasing utility and efficiency will be applied to many steps spanning this process. Chou noted the progress made by MPSbyNMR researchers in many of these areas, such as sample preparation, NMR experimentation, and structure calculation and refinement.

Understanding the interaction of MPs with detergents and lipids is integral to NMR sample preparation. Researchers led by Chuck Sanders at Vanderbilt University2kdc have studied the properties of integral membrane enzyme diacyl gylcerol kinase (DAGK; left) in detergent micelles with and without lipids. This has implications for understanding the mechanism of integral MPs as well as the use of C(14)-based detergents in future studies (Koehler, et al., 2010). In terms of increasing sensitivity of experiments for larger membrane proteins, NMR spectroscopists in the lab of Gerhard Wagner at Harvard Medical School have developed a novel 2D NMR pulse program for sequentially connecting amide resonances, hCaN (Gal, et al., 2010). This 2D pulse sequence allows researchers to tackle membrane protein complexes larger than what current methods can afford.

An overview of advances in other areas of membrane protein structure determination by NMR is given in our Featured Technology Archive: Leveraging the Power of Molecular Fragment Replacement to Solve Protein Structures by NMR. This feature introduces the work of Berardi, et al., who used site-directed paramagnetic tagging to gain long-range distance information, DNA nanotube alignment media for weakly aligning molecules in the magnetic field to determine orientation information, and experimentally determined restraints to perform molecular fragment searching and construct a model used in the structure determination of a mitochondrial proton translocator, UCP2 (Berardi, et al., 2011). “We are excited to use these techniques to obtain different functional states of the same carrier, as well as structures of different carriers, and so that we can begin to understand the biological mechanism of substrate transport,” Chou said.

Posted July 2012.

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