human ace2 (hace2) expression construct  (Gene Universal Inc)

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: (A) Infectivity quantification for HIV-1 lentivirus particles carrying various spike variants determined on hACE2 expressing 293T cells (293T-ACE2). Infectivity (mean ± s.d.) was measured from three independent experiments in triplicates. RLU, relative light units. (B) Example fluorescence trace (LD555, green; LD655, red) and resulting quantified FRET traces (FRET efficiency, blue; hidden Markov model initialization, red) of a dually labeled ligand-free spike protein on the surface of HIV-1 lentivirus particle. The single-step photobleaching step of dyes at the single-molecule level define the baseline (dashed black). Four distinguishable FRET-populated states are indicated as color-coded dash lines. ( C, D ) FRET histograms (left) and TDPs (right) of ligand-free D614 spike (S D614 , C ) and G614 spike (S G614 , D ) on lentivirus particles. A number ( N m ) of individual active/dynamic molecules - FRET traces were compiled into a conformation-population FRET histogram (gray lines) and fitted into a 4-state Gaussian distribution (solid black) centered at 0.1-FRET (dashed cyan), 0.3-FRET (dashed red), 0.5-FRET (dashed green), and 0.75-FRET (dashed magenta). TDPs show the distributions of initial and final FRET values for every observed transition in FRET traces. TDPs are displayed as initial FRET vs. final FRET with relative frequencies (max red scale = 0.01 transitions/second), originated from the idealization of individual FRET traces in FRET histograms. TDPs trace the locations of state-to-state transitions and their relative frequencies. ( E, F ) Experiments as in (C, D), respectively, conducted in the presence of 200 μg/mL monomeric hACE2. The soluble hACE2 activates spike proteins on the virus by shaping the conformational landscape toward stabilizing the “all-RBD-up” conformation (activated state). FRET histograms represent mean ± SEM, determined from three randomly assigned populations of FRET traces under corresponding experimental conditions. N m , number of individual FRET traces. Evaluated relative state occupancies see .

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques: Infection, Expressing, Fluorescence, Labeling, Virus

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: ( A, B ) FRET histograms (left) and TDPs (right) of S Alpha variant (S Alpha ) on lentivirus particles with ( A ) and without ( B ) 200 μg/mL hACE2 presence. The soluble hACE2 activates the S Alpha by shaping the conformational landscape toward stabilizing the “all-RBD-up” conformation. (C) Representative fluorescence traces (LD555, green; LD655, red) and quantified FRET traces of a single ligand-free E484K carrying S Alpha variant (S Alpha+E484K ) on lentivirus particles. (D) The FRET histogram (left) and TDP (right) of ligand-free S Alpha+E484K on lentivirus particles. ( E, F ) Experiments as in (C, D), conducted in the presence of 200 μg/mL hACE2. FRET histograms represent mean ± SEM, determined from three randomly assigned populations of all FRET traces under corresponding experimental conditions. N m , number of individual FRET traces. ( G ) Quantification of the FRET-indicated state occupancy for different spike variants. The occupancy in each FRET state was presented as mean ± SEM, determined by estimating the area under each Gaussian curve in FRET histograms. Fitting parameters see .

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques: Variant Assay, Fluorescence

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: ( A, B ) Example fluorescence traces (LD555, green; LD655, red) and resulting quantified low-FRET dominated traces (FRET efficiency, blue; hidden Markov model initialization, red) of lentivirus-embedded spikes variants G614 ( A ) and Alpha ( B ) in response to hACE2.

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: ( A, B ) Survival probability plots of four FRET-indicated conformations of lentivirus particles-associated spike proteins S D614 ( A ) and S G614 ( B ) in the absence (ligand-free, top row) and the presence of 200 μg/ml hACE2 (bottom row). The survival probability plot is the time-duration distribution of spikes dwelling on one conformation before directionally transition to the other. Transition rates (summarized in ) were estimated by double exponential-fitting of survival probability plots.

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques:

Journal: bioRxiv

Article Title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

doi: 10.1101/2021.10.11.463956

Figure Lengend Snippet: ( A - D ) Free-energy models of parental S D614 ( A ) and variants S G614 ( B ), S Alpha ( C ), and S Alpha+E484K ( D ). The differences in free energies between states were roughly scaled based upon the relative state occupancies of each state. The ligand-free D614G and E484K carrying spikes are dominated by the intermediate-FRET state (“one/two-RBD-up”), which exhibit the lowest relative free energy among all four FRET states. In contrast, all hACE2-bound spikes show the lowest relative free energy at the low-FRET state (“all-RBD-up”).

Article Snippet: The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors.

Techniques: