Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination

Abstract

The human chemokine receptor CXCR1 is a G-protein coupled receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with 13C and 15N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35 °C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35 °C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.

Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples

“…Final proteoliposome samples contain virtually no detergent. If residual detergent is suspected, evaporative light-scattering technology coupled with HPLC 39 can be used to monitor the presence of detergent in the sample. For the samples exemplified here, the concentration of detergent was below the limit of detection and is many orders of magnitude less than the protein concentration.…”

Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor

“…Fusion protein tags have proved to be instrumental in heterologous expression and purification of recombinant proteins (Sorensen and Mortensen 2005;Walls and Loughran 2011). In many instances, tags such as Glutathione S-transferase (Park et al 2012a) and maltose-binding protein (Buck et al 2003) have been used enhance the expression and facilitate purification. These, however, are sizable proteins that must be removed before subsequent study.…”

Recent Advances in Magic‐Angle Spinning Solid‐State NMR of Proteins

“…[220] Distance constraints determined by SSNMR spectroscopy facilitated the determination of the high-resolution structure of the TTR(105-115) monomer, [71] and constrained the interstrand and intersheet arrangements. TTR (105)(106)(107)(108)(109)(110)(111)(112)(113)(114)(115) peptides were arranged into parallel, in-register b-sheets stacked antiparallel to one another (Figure 8). …”

“…[95,99,100] Such labeling is more sparse than that of glycerol-based approaches, which further reduces dipolar truncation effects and facilitates distance measurements. [101] Although multiple attempts have been made to express and isotopically label G-protein coupled receptors (GPCRs) in E. coli, [102][103][104][105] with one resulting in the highresolution structure of the CXCR1 receptor, [14] the overexpression of eukaryotic proteins in E. coli is generally a difficult task. Among alternative approaches, the yeast Pichia pastoris is one of the most commonly used hosts for high-level expression of proteins that misfold in E. coli, or require post-translational modifications (PTM) for proper function.…”