Structural studies of large integral membrane proteins in reverse micelles by solution nuclear magnetic resonance

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Doctor of Philosophy (PhD)
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Biochemistry & Molecular Biophysics
nuclear magnetic resonance
reverse micelles
membrane proteins
potassium channels
Biological and Chemical Physics
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The structural characterization of integral membrane proteins represents one of the many challenges of the post-genomic era. While membrane proteins comprise approximately 50% of current and potential drug targets, their structural characterization lags far behind that of soluble proteins. Nuclear magnetic resonance (NMR) offers tremendous potential for the investigation of membrane proteins in aqueous environments with respect to structural characterization, relaxation properties, and the details of small ligand interactions. However, the size limitations of solution NMR due to the slow tumbling problem have restricted comprehensive structural characterization of membrane protein NMR structures to the relatively small β-barrel proteins or helical proteins of simple topology. Here we detail an approach for the encapsulation of integral membrane proteins in reverse micelles, allowing for their study in low viscosity solvents and thus limiting the slow tumbling issue. This approach obviates the traditional compromises in sample preparation for large proteins in NMR. Using a 54 kDa construct of the homotetrameric potassium channel KcsA, we present a hybrid surfactant screen to optimize NMR conditions and describe utilization of 3D NMR pulse sequences and backbone assignment strategies normally restricted to proteins of much smaller size. We are able to confirm the helical structure of KcsA’s transmembrane domains in reverse micelles, as well as proper quaternary arrangement of the monomers and preservation of potassium coordination in the selectivity filter. Additionally we show that the solvation properties of the channel in reverse micelles are analogous to a membrane protein solubilized by a traditional aqueous micelle. Relaxation studies of the channel are also presented.

Wand, A. Joshua
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