Abstract
The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.
Highlights
A central feature of biological activity is the correct folding of proteins into their functional states
The change in tryptophan fluorescence was monitored during denaturation, as an increase in exposure of tryptophan residues to water is accompanied by a red-shift in the fluorescence emission maximum to longer wavelengths
We address a fundamental facet of G protein coupled receptors (GPCRs) function, their folding, and report the functional folding of the exemplary GPCR, β1AR-m23, from a chemically-denatured state
Summary
A central feature of biological activity is the correct folding of proteins into their functional states. The lack of in vitro folding information on this particular protein class has significant ramifications It impacts directly upon achieving and maintaining a correctly folded, functional structure for the plethora of biophysical, biochemical and structural work aimed at ascertaining functional mechanisms for GPCRs. Obtaining sufficient quantities of properly folded GPCRs for in vitro structural and functional studies has proven tremendously difficult, not least due to the poor long-term stability of the receptors in detergents and associated loss of structure and function. We compare refolding in detergent micelles composed of DM in bulk solution with a solid support The latter approach gives unprecedented reversible refolding of the GPCR, from a urea-denatured state, with a 100% yield. It is vital to attain complete recovery of such function as membrane proteins are notorious for perturbations of ligand binding constants and reduced stability when solubilised in detergents, even without any refolding
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