Protein-based biological drugs and many industrial enzymes are unstable, making them prohibitively expensive. Some can be stabilized by formulation with excipients, but most still require low temperature storage. In search of new, more robust excipients, we turned to the tardigrade, a microscopic animal that synthesizes cytosolic abundant heat soluble (CAHS) proteins to protect its cellular components during desiccation. We find that CAHS proteins protect the test enzymes lactate dehydrogenase and lipoprotein lipase against desiccation-, freezing-, and lyophilization-induced deactivation. Our data also show that a variety of globular and disordered protein controls, with no known link to desiccation tolerance, protect our test enzymes. Protection of lactate dehydrogenase correlates, albeit imperfectly, with the charge density of the protein additive, suggesting an approach to tune protection by modifying charge. Our results support the potential use of CAHS proteins as stabilizing excipients in formulations and suggest that other proteins may have similar potential.

Escherichia coli topoisomerases I and III (Topo I and Topo III) relax negatively supercoiled DNA and also catenate/decatenate DNA molecules containing single-stranded DNA regions. Although these enzymes share the same mechanism of action and have similar structures, they participate in different cellular processes. In bulk experiments Topo I is more efficient at DNA relaxation, whereas Topo III is more efficient at catenation/decatenation, probably reflecting their differing cellular roles. To examine the differences in the mechanism of these two related type IA topoisomerases, single-molecule relaxation studies were conducted on several DNA substrates: negatively supercoiled DNA, positively supercoiled DNA with a mismatch and positively supercoiled DNA with a bulge. The experiments show differences in the way the two proteins work at the single-molecule level, while also recovering observations from the bulk experiments. Overall, Topo III relaxes DNA efficiently in fast processive runs, but with long pauses before relaxation runs, whereas Topo I relaxes DNA in slow processive runs but with short pauses before runs. The combination of these properties results in Topo I having an overall faster total relaxation rate, even though the relaxation rate during a run for Topo III is much faster.

Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed. Although recent work has shed light on the structure of the LPL monomer, the inactive oligomer remained opaque. Here we present a cryo-EM reconstruction of a helical LPL oligomer at 3.8-Å resolution. Helix formation is concentration-dependent, and helices are composed of inactive dihedral LPL dimers. Heparin binding stabilizes LPL helices, and the presence of substrate triggers helix disassembly. Superresolution fluorescent microscopy of endogenous LPL revealed that LPL adopts a filament-like distribution in vesicles. Mutation of one of the helical LPL interaction interfaces causes loss of the filament-like distribution. Taken together, this suggests that LPL is condensed into its inactive helical form for storage in intracellular vesicles.

Topoisomerases are enzymes involved in maintaining the topological state of cellular DNA. Despite many structural, biophysical, and biochemical studies, their dynamic characteristics remain poorly understood. Recent single molecule experiments revealed that an important feature of the type IA topoisomerase mechanism is the presence of pauses between relaxation events. However, these experiments cannot determine whether the protein remains DNA bound during the pauses or the relationship between domain movements in the protein and topological changes in the DNA. By combining two orthogonal single molecule techniques, we observed that topoisomerase IA is constantly changing conformation and attempting to modify the topology of DNA, but only succeeds in a fraction of the attempts. Thus, its mechanism can be described as a series of DNA strand passage attempts that culminate in a successful relaxation event.

Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing them from triglyceride-rich lipoproteins to release free fatty acids. LPL activity is regulated in a nutritionally responsive manner by macromolecular inhibitors including angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4). However, the mechanism by which ANGPTL3 inhibits LPL is unclear, in part due to challenges in obtaining pure protein for study. We used a new purification protocol for the N-terminal domain of ANGPTL3, removing a DNA contaminant, and found DNA-free ANGPTL3 showed enhanced inhibition of LPL. Structural analysis showed that ANGPTL3 formed elongated, flexible trimers and hexamers that did not interconvert. ANGPTL4 formed only elongated flexible trimers. We compared the inhibition of ANGPTL3 and ANGPTL4 using human very-low-density lipoproteins as a substrate and found both were noncompetitive inhibitors. The inhibition constants for the trimeric ANGPTL3 (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) were only 2-fold different. Heparin has previously been reported to interfere with ANGPTL3 binding to LPL, so we questioned if the negatively charged heparin was acting in a similar fashion to the DNA contaminant. We found that ANGPTL3 inhibition is abolished by binding to low-molecular-weight heparin, whereas ANGPTL4 inhibition is not. Our data show new similarities and differences in how ANGPTL3 and ANGPTL4 regulate LPL and opens new avenues of investigating the effect of heparin on LPL inhibition by ANGPTL3.