Bisht, M. | Search bioRxiv

bioRxiv2023

Nanostructured Hydrotropes Caged Cytochrome c with Boosted Stability in Harsh Environments: A Molecular Insights

Green and nano-structured catalytic media are vital for bio-catalysis to attenuate the denaturation tendency of biocata-lysts under severe reaction conditions. Hydrotropes with multi-faceted physiochemical properties represent promising systems for sustainable protein packaging. Herein, the suitability of adenosine-5-triphosphate (ATP) and cholinium sa-licylate ([Cho][Sal]) ionic liquid (IL) to form nano-structures and to nano-confine Cytochrome c (Cyt c) were demonstrat-ed to enhance the stability and activity under multiple stressors. Experimental and computational analyses were under-taken to explain the nano-structured phenomenon of ATP and IL, structural organizations of nano-confined Cyt c, and site-specific interactions that stabilize the protein structure. Both ATP and IL form nano-structures in aqueous media and could cage Cyt c via multiple nonspecific soft interactions. Remarkably, the engineered molecular nano-cages of ATP (5-10 mM), IL (300 mg/mL), and ATP+IL surrounding Cyt c resulted in 9-to-72-fold higher peroxidase activity than native Cyt c with exceptionally high thermal tolerance (110oC). The polar interactions with the cardiolipin binding site of Cyt c, mediated by hydrotropes, were well correlated with the increased peroxidase activity. Furthermore, higher activity trends were observed in the presence of urea, GuHCl, and trypsin without any protein degradation. Specific binding of hy-drotropes in highly mobile regions of Cyt c ({Omega} 40-54 residues) and enhanced H-bonding with Lys and Arg offered excel-lent stability under extreme conditions. Additionally, ATP effectively counteracted reactive oxygen species (ROS)-induced denaturation of Cyt c, which was enhanced by the [Sal] counterpart of IL. Overall, this study explored the robustness of nano-structured hydrotropes to have a higher potential for protein packaging with improved stability and activity under extreme conditions. Thus, the present work highlights a novel strategy for real-time industrial bio-catalysis to protect mitochondrial cells from ROS-instigated apoptosis. SummarySuitability of ATP and [Cho][Sal] ionic liquid to form nanostructured hydrotropes and their utility in protein packaging in extreme conditions are discussed. Both ATP and IL form nanostructures in aqueous media and could cage Cyt c via multiple nonspecific soft interactions. The engineered molecular nanocages surrounding Cyt c resulted in 9-to-72-fold higher peroxidase activity than native Cyt c with exceptionally high thermal tolerance (110{degrees}C) and stability in the presence of urea, GuHCl, and trypsin without any protein degradation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/527166v3_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@167eb39org.highwire.dtl.DTLVardef@183fa44org.highwire.dtl.DTLVardef@1a38f57org.highwire.dtl.DTLVardef@117d203_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

CRISPR-based multi-locus real-time tracking reveals single chromosome dynamics and compaction

In eukaryotic nuclei, individual chromosomes occupy discrete three-dimensional spaces with little overlap. Dynamic chromatin organization instantly influences DNA accessibility through modulating local macromolecular density and interactions, driving changes in transcription activities. Chromatin anchoring to nuclear landmarks often leads to massive reorganization and motion changes. Chromatin dynamics has been reported to be locally confined but contributes to the large-scale coherent chromatin motion across the entire nucleus. However, the dynamics and compaction of chromosomal sub-regions along a single chromosome are not well-understood. In this study, we combined quantitative real-time single-molecule fluorescence microscopy, CRISPR-based genomic labeling, biophysical analysis and polymer models to characterize the dynamics of specific genomic loci and chromatin-nuclear landmark interactions on human chromosome 19 in living cells. Precise genomic labeling allows us to dissect loci motions and chromatin elasticity on a single chromosome basis. We found that the dynamics of genomic loci were all subdiffusive but varied at different regions along the chromosome. The mobility of genomic loci was similar among interior chromosomal loci but deviated for the loci at pericentromeric and near-telomeric regions. Tighter compaction on chromosome 19 long arm, compared to the short arm was observed, which may correlate to more active genes on the short arm than the long arm, shown by our RNA-seq analysis. The strong tethering interaction was found for loci at the pericentromeric region, suggesting a higher degree of local condensation, perhaps through stronger interactions or association between pericentromeric regions and their microenvironments, such as chromatin-nuclear body association through sequence-specific domains on DNA.

biophysics↗