For a typical (20 kD, 4 nm size) monomeric enzyme, more than 50% of the residues are at the surface. The mechanics of these soft, heterogeneous nanoparticles was recently shown to be viscoelastic. Here, we explore the contribution of the enzyme's surface to the mechanics of the molecule. Nano-rheology provides sub-Å resolution measurements of the reversible deformation of the enzyme subject to an oscillatory mechanical stress. We perturb the surface of the enzyme by adding small amounts of DMSO (dimethyl sulfoxide), believed to affect ordering of the enzyme–water interface. We observe a dramatic though reversible change in the mechanics of the enzyme, which becomes more viscous. On the other hand, the catalytic speed is unaffected, while at higher DMSO concentrations (>1 %) it even increases.
Chemical agents are classified into chaotropes (disorder inducing) and kosmotropes (order inducing) based on their ability to destabilize or stabilize, respectively, the structure of hydrated macromolecules and their solutions. Here, we examine the effect of such agents on the mechanical stiffness of the hydration layer of proteins, measured by nanorheology. We examine four different agents and conclude that chaotropic substances induce the overall softening of the protein-hydration layer system, whereas the kosmotropic substances induce stiffening. Specifically, with glucose and trifluoroethanol, two known kosmotropic agents, we observe the stiffening of the hydration layer. In contrast, with guanidine hydrochloride and urea, known kaotropic agents, we observe softening. Thus, the viscoelastic mechanics of the folded, hydrated protein provides an experimental measure of the effect that chaotropes and kosmotropes have on the dynamics.
Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (K ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.
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