Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures ( <1 Å), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade‐unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS‐8 from Hypsibius exemplaris. NMR spectroscopy reveals that the protein comprises an extended central helical domain flanked by disordered termini. Upon concentration, the protein is shown to successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature‐dependent manner. The helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel. Soluble proteins can be encapsulated within cavities in the gel, maintaining their functional form. The ability to reversibly form fibrous gels may be associated with the enhanced protective properties of these proteins.

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade‐unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS‐8 from Hypsibius exemplaris. NMR spectroscopy reveals that the protein comprises an extended central helical domain flanked by disordered termini. Upon concentration, the protein is shown to successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature‐dependent manner. The helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel. Soluble proteins can be encapsulated within cavities in the gel, maintaining their functional form. The ability to reversibly form fibrous gels may be associated with the enhanced protective properties of these proteins.

We have extended the finite-difference Poisson-Boltzmann (FDPB) equation method to incorporate the treatment of mixed salts (i.e. NaCl/MgCl 2 ). In this context, we have derived an expression for the total electrostatic free energy for mixed salt systems. We use the theory to study nonspecific mixed salt effects on the binding free energies of the minor groove binding antibiotic DAPI, and λ repressor, with DNA. We find that in a pure salt solution the electrostatic contribution to binding varies linearly with log[M n+ ], (where M n+ represents an n-valent cation) and that the effect is uncorrelated with either the valence of the binding ligand or the number of counterions in the binding site. In mixed salt solution, the monovalent and divalent counterions "compete" for the immediate vicinity of the DNA. As experimentally observed in mixed salt solutions, a pronounced curvature appears in the plot of the electrostatic binding free energy vs log[M n+ ]. The curvature for DAPI-DNA binding in mixed salts reflects the fact that divalent counterions interact with bound or free DNA molecules more strongly than monovalent counterions. However, the valence dependence of the electrostatic interaction between cations and negatively charged macromolecules is not solely responsible for the observed curvature. Rather anions, which can interact quite strongly with highly charged DNAbinding proteins, make a significant contribution to the observed salt effects. Our results support previous findings that treatments based on counterion condensation concepts break down for protein-DNA interactions; specifically, it is necessary to describe molecular structures in atomic detail if a realistic description of salt effects is to be obtained.