High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination.

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (that is, unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here, we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have dissociation constant Kd values down to 60 pM and, together with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.

Caenorhabditis elegans Hsp70-1 expresses highly in bacteria, is sufficiently soluble, and has a catalytic constant similar to Hsc70 and BiP.

Caenorhabditis elegans has been used as a model organism to study the roles of molecular chaperones in cellular processes. C. elegans heat shock protein 70-1 (CeHsp70-1) is the first of the 13-member Hsp70 family genes identified so far in the organism. The protein product of this gene, CeHsp70-1, has been shown to play an important role in conferring thermo-tolerance and longevity on C. elegans. Here, we present the results of the first work to over-express, purify and characterize the ATP hydrolyzing activity of a member of the C. elegans Hsp70s. Recombinant CeHsp70-1 was found to be highly expressed and sufficiently soluble in Escherichia coli. The protein was purified to homogeneity using a combination of nickel affinity, ion exchange and size-exclusion chromatography. Kinetic properties of the basal ATPase activity of the enzyme in a low-salt buffer were determined using a colorimetric assay. The specific activity (V(max) per mg protein), K(m) and k(cat) values obtained for CeHsp70-1 were 25 nmol/min/mg, 50 µM and 0.28 min⁻¹, respectively. The catalytic constant (k(cat)) of the protein was found to be similar to that of heat shock cognate 70 (Hsc70) and binding immunoglobulin protein (BiP). At low concentrations, CeHsp70-1 existed mostly in its monomeric form. This work provides a platform for kinetic studies of other members of the C. elegans Hsp70 molecular chaperones.