Potassium Channel KcsA and Its Lipid Environment

There is a general lack of atomic resolution data of mobile regions of membrane proteins embedded in lipid bilayers. As an inherently complex system, few techniques can capture information about the mobile portions of an otherwise immobilized protein. The nature of crystallography and solid-state NMR relies on structural rigidity. Solution-state NMR relies on overall mobility of a protein for resolution. In the middle regime, there are few solutions to study these systems. The inward-rectifying, pH-gated potassium channel KcsA from Streptomyces lividans makes an excellent model for the development of methods to study mobile regions of membrane proteins. Of its 160 residues, more than a third are in extracellular do- mains and are not typically captured by solid-state NMR or crystallographic techniques. These pages present evidence that KcsA’s C-terminus is highly mobile and becomes increasingly dynamic when the protein is at low pH and high K+ concen- tration, where the channel is known to be active. By applying proton-detected, high-resolution magic angle spinning NMR (HR-MAS) to fractionally deuterated KcsA, previously unattainable correlations are collected and new resonance assignments are made, demonstrating the utility of the technique. The lipid environment is well known to regulate the function of KcsA in particular and membrane proteins in general. It is generally assumed that reconstituting KcsA into a synthetic phospholipid membranes provides the protein a well-defined environment. Data is presented here which shows that KcsA co-purifies with phosphoglycerol lipids from the E. coli membrane and that these molecules are 13C enriched in the course of isotopically labeling KcsA. Further, significant hydrolysis of both co- purifying and synthetic lipids occurs under ordinary experimental conditions. These findings demand that routine analysis of samples must include verification of the chemical integrity of lipids. Finally, the feasibility of applying dynamic nuclear polarization-enhanced NMR (DNP) to KcsA is investigated as a means of elucidating information about its termini. Although KcsA is known to enhance poorly by DNP, data presented here show that this is not an intrinsic property of the protein but rather an effect of the matrix in which KcsA is investigated. The use of a 15N-enriched free amino acid dissolved into buffers used for DNP is shown to be a powerful diagnostic internal standard