Fig. 5: Cellular expression, thermostability, and structural characterization of HexaPro-SS-2W.
From: Simulation-driven design of stabilized SARS-CoV-2 spike S2 immunogens
a SDS-PAGE of purified S2 constructs (Base, HexaPro-SS-V991W, HexaPro-SS-T998W, and HexaPro-SS-2W). The ‘Base’ construct corresponds to HexaPro-SS-∆stalk. No statistical analysis was performed on the SDS-PAGE gel. The uncropped gel image is included in the Source Data file. b Size-exclusion chromatography (SEC) of purified S2 constructs. Both tryptophan substitutions increase protein expression yield, with V991W + T998W and T998W resulting in a greater increase. c Differential scanning fluorimetry of S2 constructs, including the original HexaPro (S1 + S2). HexaPro-SS-2W exhibits superior thermal stability to all HexaPro constructs, with a ~16 °C increase in Tm relative to HexaPro. d Cryo-EM map (Resolution: 2.8 Å) of closed prefusion state HexaPro-SS-2W (∆stalk) from side and top-down perspectives. e–g Top-down perspective of HexaPro-SS-2W structure highlighting V991W (orange) and T998W (red) packing within the S2 interior; the tryptophan sidechains self-associate between chains in offset edge-to-edge and edge-to-face π–π stacking orientations. The EM map is shown as a transparent gray volume.
