Cavity-based free energy analysis of osmolyte effects on protein denaturation.

Experimental measurements of the thermal effects of the same osmolytes on two different globular proteins, C-reactive protein (CRP) and tumor necrosis factor alpha (TNFα), have shown that quantifying the change in the denaturing temperature leads to some results that are unique to each protein. In order to find osmolyte-dependent parameters that can be applied more consistently from protein to protein, this work considers, instead, the overall free energy change associated with that denaturation using coarse-grained models. This is enabled by using theoretical fluid equations that take into account the exclusion of water and osmolyte from the volume occupied by the protein in both its native and denatured forms. Assuming ideal geometric models of the two protein states whose sizes are based on the protein's surface area in each form, and taking into account the density of the aqueous osmolyte solution, the free energy change due to the change in geometry can be calculated. The overall change in free energy of the system is found from that quantity and other protein- and osmolyte-specific parameters, which are determined using the experimental concentration and temperature results. We find that these fitted parameters accurately reproduce experimental results and also show consistent patterns from protein to protein. We also consider two different model geometries of the denatured protein and find little impact on the use of one or the other. Defining the effects of the osmolyte in terms of free energy also allows for prediction of overall phase change behavior, including cold denaturation.

Synthesis and Structure-Activity Relationship of Dual-Stage Antimalarial Pyrazolo[3,4-b]pyridines.

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.

Hybrid Ptr2-like activators of archaeal transcription.

Methanocaldococcus jannaschii Ptr2, a member of the Lrp/AsnC family of bacterial DNA-binding proteins, is an activator of its eukaryal-type core transcription apparatus. In Lrp-family proteins, an N-terminal helix-turn-helix DNA-binding and dimerizing domain is joined to a C-terminal effector and multimerizing domain. A cysteine-scanning surface mutagenesis shows that the C-terminal domain of Ptr2 is responsible for transcriptional activation; two types of DNA binding-positive but activation-defective mutants are found: those unable to recruit the TBP and TFB initiation factors to the promoter, and those failing at a post-recruitment step. Transcriptional activation through the C-terminal Ptr2 effector domain is exploited in a screen of other Lrp effector domains for activation capability by constructing hybrid proteins with the N-terminal DNA-binding domain of Ptr2. Two hybrid proteins are effective activators: Ptr-H10, fusing the effector domain of Pyrococcus furiosus LrpA, and Ptr-H16, fusing the P. furiosus ORF1231 effector domain. Both new activators exhibit distinguishing characteristics: unlike octameric Ptr2, Ptr-H10 is a dimer; unlike Ptr2, the octameric Ptr-H16 poorly recruits TBP to the promoter, but more effectively co-recruits TFB with TBP. In contrast, the effector domain of Ptr1, the M. jannaschii Ptr2 paralogue, yields only very weak activation.