The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular-dynamics simulations

M. Caldarini, P. Sonar, I. Valpapuram, D. Tavella, C. Volonte, V. Pandini, M.A. Vanoni, A. Aliverti, R.A. Broglia, G. Tiana, C. Cecconic

We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Under our experimental conditions the PF state is quite compact, its extension being essentially indistinguishable from that of the N state. Refolding of mechanically-denatured HIV-1-PR monomers is a multiple-pathway process, where the protein reaches its native state through different trajectories. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, which differ for number and type of non-native contacts, and provide information regarding possible structural features of the PF state.

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