Abstract
Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies.
Highlights
Integral membrane proteins (MP) constitute around 30% of the proteome [1] and they are fundamental for homeostatic maintenance of living cells, which involves a controlled flow of water and substrates across cellular membranes and signal transduction through MP receptors
We applied bioinformatics to quantify the occurrence of N- and Cterminal intrinsically disordered regions (IDR) in human MPs
We find that ~22% of the MPs do not have a disordered N-terminal longer than five residues (Fig. 2A) and ~51% do not have a disordered C-terminal longer than five residues (Fig. 2B)
Summary
Integral membrane proteins (MP) constitute around 30% of the proteome [1] and they are fundamental for homeostatic maintenance of living cells, which involves a controlled flow of water and substrates across cellular membranes and signal transduction through MP receptors. Prokaryotic protein, which are easier to purify due to their shorter extramembrane regions, have been used extensively to model human MPs [7]. This approach is limited by the fact that around 85% of human MPs lack prokaryotic counterparts [8]. Single pass receptors and multi-spanning MPs, like transporters, pumps and channels, play pivotal roles both physiologically and in pathophysiology, and represent an understudied area in structural biology. This limits development of drugs targeting these MP classes [11].
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