9 research outputs found
Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes
The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of âpublicâ antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that âpublicâ NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape
A Humanized CB1R Yeast Biosensor Enables Facile Screening of Cannabinoid Compounds
Yeast expression of human G-protein-coupled receptors (GPCRs) can be used as a biosensor platform for the detection of pharmaceuticals. Cannabinoid receptor type 1 (CB1R) is of particular interest, given the cornucopia of natural and synthetic cannabinoids being explored as therapeutics. We show for the first time that engineering the N-terminus of CB1R allows for efficient signal transduction in yeast, and that engineering the sterol composition of the yeast membrane modulates its performance. Using an engineered cannabinoid biosensor, we demonstrate that large libraries of synthetic cannabinoids and terpenes can be quickly screened to elucidate known and novel structureâactivity relationships. The biosensor strains offer a ready platform for evaluating the activity of new synthetic cannabinoids, monitoring drugs of abuse, and developing therapeutic molecules
Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration
Understanding protein stability is
central to combatting protein
aggregation diseases and developing new protein therapeutics. At the
high concentrations often present in biological systems, purified
proteins can exhibit undesirable high solution viscosities and poor
solubilities mediated by short-range electrostatic and hydrophobic
proteinâprotein interactions. The interplay between protein
amino acid sequence, protein structure, and solvent conditions to
minimize proteinâprotein interactions is key to designing well-behaved
pharmaceutical proteins. However, theoretical approaches have yet
to yield a general framework to address these problems. Here, we analyzed
the high concentration behavior of superfolder GFP (sfGFP) and two
supercharged sfGFP variants engineered to have formal charges of â18
or +15. Under low cosolute conditions, sfGFP and the â18 variant
formed a gel or phase separated at âŒ10 mg/mL. Under conditions
that screen surface charges, including formulations with high histidine
or high NaCl concentrations, all three variants attained concentrations
up to 250 mg/mL with moderate viscosities. Moreover, all three variants
exhibited very similar viscosityâconcentration profiles over
this range. This effect was not mimicked by high sugar concentrations
that exert excluded-volume effects without shielding charge. Collectively,
these data demonstrate that charge shielding neutralizes not only
long-range electrostatic interactions but also, surprisingly, short-range
electrostatic effects due to surface charge anisotropy. This work
shows that supercharged sfGFP behavior under high ionic strength is
largely determined by particle geometry, a conclusion that is supported
by colloid models and may be applicable to pharmaceutically relevant
proteins