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Archaeal group II chaperonins, also known as heat shock proteins (HSPs), are abundantly expressed in Sulfolobales. HSPα and HSPβ gene expression is upregulated during thermal shock. HSPs form large 18-mer complexes that assist in folding nascent proteins and protecting resident proteins during thermal stress. Engineered HSPs have been designed for industrial applications. Since temperature flux in the geothermal habitats of Sulfolobales impacts intracellular temperature, it follows that HSPs have developed thermotolerance. However, despite the low pH (i.e., pH < 4) typical for these habitats, intracellular pH in Sulfolobales is maintained at ~6.5. Therefore, it is not presumed that HSPs have evolved acid-tolerance. To test tolerance to low pH, HSPs were studied at various pH and temperature values. Both circular dichroism and intrinsic fluorescence indicate that HSPα and HSPβ retain structural integrity at neutral pH over a wide range of temperatures. Structural integrity is compromised for all HSPs at ultra-low pH (e.g., pH 2). Secondary structures in HSPs are resilient under mildly acidic conditions (pH 4) but Anilino naphthalene 8-sulfonate binding shows shifts in tertiary structure at lower pH. Trypsin digestion shows that the HSPβ-coh backbone is the most flexible and HSPβ is the most resilient. Overall, results suggest that HSPα and HSPβ exhibit greater thermostability than HSPβ-coh and that there are limits to HSP acid-tolerance. Molecular dynamics (MD) simulations complement the wet lab data. Specifically, MD suggests that the HSPβ secondary structure is the most stable. Also, despite similarities in pH- and temperature-dependent behavior, there are clear differences in how each HSP subtype is perturbed. 

Abstract The protein–ligand binding affinity quantifies the binding strength between a protein and its ligand. Computer modeling and simulations can be used to estimate the binding affinity or binding free energy using data- or physics-driven methods or a combination thereof. Here we discuss a purely physics-based sampling approach based on biased molecular dynamics simulations. Our proposed method generalizes and simplifies previously suggested stratification strategies that use umbrella sampling or other enhanced sampling simulations with additional collective-variable-based restraints. The approach presented here uses a flexible scheme that can be easily tailored for any system of interest. We estimate the binding affinity of human fibroblast growth factor 1 to heparin hexasaccharide based on the available crystal structure of the complex as the initial model and four different variations of the proposed method to compare against the experimentally determined binding affinity obtained from isothermal titration calorimetry experiments. 

Abstract Human acidic fibroblast growth factor (hFGF1) is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding. In this study, we investigate the equilibrium unfolding of hFGF1 using a wide array of biophysical and biochemical techniques. Systematic analyses of the thermal and chemical denaturation data on hFGF1 variants (Q54P, K126N, R136E, K126N/R136E, Q54P/K126N, Q54P/R136E, and Q54P/K126N/R136E) indicate that nullification of charges in the heparin-binding pocket can significantly increase the stability of wtFGF1. Triple variant (Q54P/K126N/R136E) was found to be the most stable of all the hFGF1 variants studied. With the exception of triple variant, thermal unfolding of wtFGF1 and the other variants is irreversible. Thermally unfolded triple variant refolds completely to its biologically native conformation. Microsecond-level molecular dynamic simulations reveal that a network of hydrogen bonds and salt bridges linked to Q54P, K126N, and R136E mutations, are responsible for the high stability and reversibility of thermal unfolding of the triple variant. In our opinion, the findings of the study provide valuable clues for the rational design of a stable hFGF1 variant that exhibits potent wound healing properties.