SARS-CoV-2 emergent variants are characterized by increased transmissibility and each show multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track correlative changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in Omicron BA.1 S traced through Alpha S and Delta S variants. Stabilization preceded progressive enhancement in dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from specific S mutations detail the evolutionary trajectory of S protein in emerging variants. These carry major implications for SARS- CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.

SARS-CoV-2 emergent variants are characterized by increased viral fitness and each shows multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in subsequent variants. Stabilization preceded altered dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from S mutations detail the evolutionary trajectory of S in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.

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BipA is a conserved translational GTPase necessary for securing bacterial survival and successful invasion of the host. Structural and biochemical studies indicate that GTP and ppGpp compete for binding to BipA to promote differential association of BipA to either the 70S or 30S ribosomal species. Exactly how guanine nucleotide binding to BipA prompts a change in the association of this protein with the ribosome is not understood. Crystallographic models show local structural rearrangements occur near the nucleotide‐binding pocket but unexpectedly the overall domain arrangement, and therefore intramolecular contacts are similar in the various guanine nucleotide bound states. One explanation is that the lattice contacts in the crystal restrict the conformational space available to the protein. Another is that the GTP‐ and ppGpp‐bound forms of BipA are dynamic meta‐stable or intermediate states poised to bind the ribosome. This model would account for a reverse flow of information evidenced by the increase in BipA’s rate of GTP hydrolysis upon ribosome binding. BipA’s hydrodynamic radius as measured by analytical ultracentrifugation and heat capacity changes assessed by isothermal titration calorimetry experimentally support our hypothesis that limited domain movements occur in solution upon guanine nucleotide binding. MD simulations utilizing the conformational end‐states provided by our GTP‐ and ppGpp‐bound crystal structures, coupled to a description of the solution dynamics of BipA using amide hydrogen/deuterium exchange mass spectrometry (HDX‐MS) indicate that this protein exists in an ensemble of states in solution as a metastable entity. The mutually exclusive binding of GTP or ppGpp produce unique allosteric relays within the protein directing the equilibria to alternate distinct conformations resulting in the formation of the two BipA:ribosome complexes. This intrinsic conformational fluidity is the origin of BipA’s ability to respond to ever changing environmental conditions synchronizing them with cellular physiology facilitating adaptation and long‐term survival of the bacteria.