Protective roles of highly conserved motif 1 in tardigrade cytosolic-abundant heat soluble protein in extreme environments.

Tardigrades are remarkable microscopic animals that survive harsh conditions such as desiccation and extreme temperatures. Tardigrade-specific intrinsically disordered proteins (TDPs) play an essential role in the survival of tardigrades in extreme environments. Cytosolic-abundant heat soluble (CAHS) protein, a key TDP, is known to increase desiccation tolerance and to protect the activity of several enzymes under dehydrated conditions. However, the function and properties of each CAHS domain have not yet been elucidated in detail. Here, we aimed to elucidate the protective role of highly conserved motif 1 of CAHS in extreme environmental conditions. To examine CAHS domains, three protein constructs, CAHS Full (1-229), CAHS ∆Core (1-120_184-229), and CAHS Core (121-183), were engineered. The highly conserved CAHS motif 1 (124-142) in the CAHS Core formed an amphiphilic α helix, reducing the aggregate formation and protecting lactate dehydrogenase activity during dehydration-rehydration and freeze-thaw treatments, indicating that CAHS motif 1 in the CAHS Core was essential for maintaining protein solubility and stability. Aggregation assays and confocal microscopy revealed that the intrinsically disordered N- and C-terminal domains were more prone to aggregation under our experimental conditions. By explicating the functions of each domain in CAHS, our study proposes the possibility of using engineered proteins or peptides derived from CAHS as a potential candidate for biological applications in extreme environmental stress responses.

Controlling the Chameleonic Behavior and Membrane Permeability of Cyclosporine Derivatives via Backbone and Side Chain Modifications.

Some macrocycles exhibit enhanced membrane permeability through conformational switching in different environmental polarities, a trait known as chameleonic behavior. In this study, we demonstrate specific backbone and side chain modifications that can control chameleonic behavior and passive membrane permeability using a cyclosporin O (CsO) scaffold. To quantify chameleonic behavior, we used a ratio of the population of the closed conformation obtained in polar solvent and nonpolar solvent for each CsO derivative. We found that ß-hydroxylation at position 1 (1 and 3) can encode chameleonicity and improve permeability. However, the conformational stabilization induced by adding an additional transannular H-bond (2 and 5) leads to a much slower rate of membrane permeation. Our CsO scaffold provides a platform for the systematic study of the relationship among conformation, membrane permeability, solubility, and protein binding. This knowledge contributes to the discovery of potent beyond the rule of five (bRo5) macrocycles capable of targeting undruggable targets.

NMR Structure and Biophysical Characterization of Thermophilic Single-Stranded DNA Binding Protein from Sulfolobus Solfataricus.

Proteins from Sulfolobus solfataricus (S. solfataricus), an extremophile, are active even at high temperatures. The single-stranded DNA (ssDNA) binding protein of S. solfataricus (SsoSSB) is overexpressed to protect ssDNA during DNA metabolism. Although SsoSSB has the potential to be applied in various areas, its structural and ssDNA binding properties at high temperatures have not been studied. We present the solution structure, backbone dynamics, and ssDNA binding properties of SsoSSB at 50 °C. The overall structure is consistent with the structures previously studied at room temperature. However, the loop between the first two ß sheets, which is flexible and is expected to undergo conformational change upon ssDNA binding, shows a difference from the ssDNA bound structure. The ssDNA binding ability was maintained at high temperature, but different interactions were observed depending on the temperature. Backbone dynamics at high temperature showed that the rigidity of the structured region was well maintained. The investigation of an N-terminal deletion mutant revealed that it is important for maintaining thermostability, structure, and ssDNA binding ability. The structural and dynamic properties of SsoSSB observed at high temperature can provide information on the behavior of proteins in thermophiles at the molecular level and guide the development of new experimental techniques.

Resonance assignments and secondary structure of thermophile single-stranded DNA binding protein from Sulfolobus solfataricus at 323K.

Single-stranded DNA (ssDNA)-binding proteins (SSBs) are essential for DNA replication, recombination, and repair processes in all organisms. Sulfolobus solfataricus (S. solfataricus), a hyperthermophilic species, overexpresses its SSB (S. solfataricus SSB (SsoSSB)) to protect ssDNA during DNA metabolisms. Even though the crystal structure of apo SsoSSB and its ssDNA-bound solution structure have been reported at room temperature, structural information at high temperature is not yet available. To find out how SsoSSB maintains its structure and ssDNA binding affinity at high temperatures, we performed multidimensional NMR experiments for SsoSSB at 323K. In this study, we present the backbone and side chain chemical shifts and predict the secondary structure of SsoSSB from the chemical shifts. We found that SsoSSB is ordered, even at high temperatures, and has the same fold at high temperature as at room temperature. Our data will help improve structural analyses and our understanding of the features of thermophilic proteins.