Effects of Hydrostatic Pressure on the Thermodynamics of CspB-Bs Interactions with the ssDNA Template

Abstract

Understanding the thermodynamic mechanisms of adaptation of biomacromolecules to high hydrostatic pressure can help shed light on how piezophilic organisms can survive at pressures reaching over 1000 atmospheres. Interaction of proteins with nucleic acids is one of the central processes that allow information flow encoded in the sequence of DNA. Here, we report the results of a study on the interaction of cold shock protein B from Bacillus subtilis (CspB-Bs) with heptadeoxythymine template (pDT7) as a function of temperature and hydrostatic pressure. Experimental data collected at different CspB-Bs:pDT7 ratios were analyzed using a thermodynamic linkage model that accounts for both protein unfolding and CspB-Bs:pDT7 binding. The global fit to the model provided estimates of the stability of CspB-Bs, ΔG Prot o , the volume change upon CspB-Bs unfolding, ΔV Prot , the association constant for CspB-Bs:pDT7 complex, K a o , and the volume changes upon pDT7 single-stranded DNA (ssDNA) template binding, ΔV Bind . The protein, CspB-Bs, unfolds with an increase in hydrostatic pressure (ΔV Prot < 0). Surprisingly, our study showed that ΔV Bind < 0, which means that the binding of CspB-Bs to ssDNA is stabilized by an increase in hydrostatic pressure. Thus, CspB-Bs binding to pDT7 represents a case of linked equilibrium in which folding and binding react differently upon an increase in hydrostatic pressure: protein folding/unfolding equilibrium favors the unfolded state, while protein−ligand binding equilibrium favors the bound state. These opposing effects set a "maximum attainable" pressure tolerance to the protein−ssDNA complex under given conditions.

Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View

“…Pressure dependent measurements of the interaction of cold-shock protein CspB-Bs binding to a pDT7 single-stranded DNA template by Avagyan and Makhatadze revealed negative volume changes upon binding (Δ V bind ≈ −10 mL mol –1 at 37 °C, decreasing in magnitude with decreasing temperature), indicating that an increase in hydrostatic pressure stabilizes CspB-Bs:pDT7b complex formation. However, due to a negative volume change of unfolding, HHP destabilizes the structure of the protein (Δ V unf = −40 mL mol –1 ) . Addition of the stabilizing osmolyte glutamate increased both the binding affinity and the stability of the protein …”

“…However, due to a negative volume change of unfolding, HHP destabilizes the structure of the protein (ΔV unf = −40 mL mol −1 ). 214 Addition of the stabilizing osmolyte glutamate increased both the binding affinity and the stability of the protein. 214…”

“…214 Addition of the stabilizing osmolyte glutamate increased both the binding affinity and the stability of the protein. 214…”

Modulation of Electrostatic Interactions as a Mechanism of Cryptic Adaptation ofColwelliato High Hydrostatic Pressure

“…We have also shown that the increase in pressure stabilizes interactions of CspB proteins with the ssDNA, i.e. the volume change upon CspB:ssDNA binding is positive [65]. Briefly, the following linked equilibrium is modulated by an increase in pressure (knowing that the ligand does not bind to the unfolded state):…”

“…Thus, the overall bound fraction (i.e. [NA:P]) as a function of the increase in pressure is largely controlled by the protein stability [65]. Considering that the DNA binding interface of CspD proteins is almost identical to that of CspB proteins, it is reasonable to assume that to maintain biological function, which is a nucleic acid binding [46][47][48]68], the protein needs to be more stable at high pressure [65].…”