Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support

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

Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in four infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein with corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ and/or TNF-ɑ producing T cells is comparable between infants and adults. On principal component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries

In Vitro Folding and Assembly of the Escherichia coli ATP-binding Cassette Transporter, BtuCD*

Studies on membrane protein folding have focused on monomeric α-helical proteins and a major challenge is to extend this work to larger oligomeric membrane proteins. Here, we study the Escherichia coli (E. coli) ATP-binding cassette (ABC) transporter that imports vitamin B12 (the BtuCD protein) and use it as a model system for investigating the folding and assembly of a tetrameric membrane protein complex. Our work takes advantage of the modular organization of BtuCD, which consists of two transmembrane protein subunits, BtuC, and two cytoplasmically located nucleotide-binding protein subunits, BtuD. We show that the BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis were achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter. BtuCD folding also provides an opportunity to investigate how a protein that contains both membrane-bound and aqueous subunits coordinates the folding requirements of the hydrophobic and hydrophilic subunits

β₁AR-m23 refolding on a Ni²⁺-NTA column into DM, from urea.

<p>4.5 μM β₁AR-m23, bound to a Ni²⁺-NTA column, was unfolded in 25 mM Tris pH 7.5, 150 mM NaCl, 0.5% DM and 8 M urea for 5 mins before refolding into buffer in which the urea had been omitted. The (a) binding of antangonist [³H]DHA to as well as the (b) fluorescence emission maximum and (d) CD signal at 222 nm of the refolded receptor. Results are compared to the originally folded β₁AR-m23 in 0.5% DM and β₁AR-m23 unfolded in 8 M urea. Error bars show ± SD and are the result of three independent experiments on different samples. (c) Fluorescence and (e) far UV-CD spectra of the original folded β₁AR-m23 in 0.5% DM (solid line) and of refolded β₁AR-m23 in 0.5% DM (dotted line). Spectra are the average of three independent measurements. The fluorescence emission maximum was 330.7 ± 0.5 nm for the original folded receptor and 331.7 ± 0.7 nm for the receptor refolded in DM. The CD signal at 222 nm was– 21520 ± 2840 deg.cm².dmol^-1 for the original folded receptor and– 21360 ± 2520 deg.cm².dmol^-1.</p

β₁AR-m23 unfolding in DM and in urea.

<p>Fluorescence and far UV CD spectra (a and b, respectively) of 0.45 μM and (c and d, respectively) 4.5 μM β₁AR-m23 in DM in the original folded state in the presence of 0 M urea (solid lines) and 3 M urea (dashed lines), and unfolded in 8 M urea (dotted lines). All buffers contained 25 mM Tris pH 7.5, 150 mM NaCl, 0.1 mM EDTA and 0.5% DM. Spectra are averages from a minimum of three independent experiments on different samples. For folded protein (in 0 M urea) the band intensity at 222 nm was -26070 ± 2400 deg.cm².dmol^-1 and -27780 ± 5400 deg.cm².dmol^-1 at a protein concentration of 0.45 μM and 4.5 μM, respectively. The wavelength at the fluorescence emission maximum for folded protein was 329.9 ± 0.8 nm and 329.8 ± 0.3 nm at a protein concentration of 0.45 μM and 4.5 μM, respectively. The intensity at the fluorescence emission maximum for folded protein was 474000 ± 118000 and 2404000 ± 399000 at a protein concentration of 0.45 μM and 4.5 μM, respectively.</p

Comparing β₁AR-m23 unfolding in bulk solution and on a solid support by pulse proteolysis.

<p>Example SDS-PAGE gels of β₁AR-m23 following 3-min pulse proteolysis after a 5 or 30 min incubation in urea. Briefly, 0.45 or 4.5 μM β₁AR-m23 in either bulk solution or bound to Ni²⁺-NTA column was equilibrated with buffer containing 25 mM Tris pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.5% DM, 10 mM CaCl₂ and 3 (folding conditions) or 8 M (unfolding conditions) urea followed by digestion with 0.2 mg/ml thermolysin for 3 min. The original folded β₁AR-m23 is shown for comparison (<i>lanes β</i>_<i>1</i><i>AR-m23</i>) and molecular mass markers are indicated in kDa (<i>lanes M</i>).</p

Monitoring β₁AR-m23 unfolding in bulk solution by pulse proteolysis.

<p>0.2 mg/ml thermolysin was used to digest 0.45 μM β₁AR-m23, pre-equilibrated in buffer containing 25 mM Tris pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.5% DM, 10 mM CaCl₂ and urea (2–8 M) for 30 min, for 1 min. (a) A representative SDS-PAGE gel of β₁AR-m23 following 1-min pulse proteolysis. (b) The percentage of folded β₁AR-m23 remaining as determined by pulse proteolysis (circles), fluorescence (squares) and far-UV CD (triangles). The percentage of folded protein at each urea concentration was determined by; pulse proteolysis from the amount of undigested protein as measured by SDS-PAGE; fluorescence from the red-shift in the fluorescence emission maximum; and CD from the degree of α-helical structure as measured by the CD intensity at 222 nm. Resulting values were normalised between 0% and 100% with 100% representing the fully folded protein in DM and the 0% the partly unfolded 8 M urea state that possesses some helical content. Error bars show ± SD (standard deviation) and are the result of a minimum of three independent experiments on different samples.</p