Equilibrium unfolding

In biochemistry, equilibrium unfolding is the process of unfolding a protein or RNA molecule by gradually changing its environment, such as by changing the temperature or pressure, adding chemical denaturants, or applying force as with an atomic force microscope tip. Since equilibrium is maintained at all steps, the process is reversible (equilibrium folding). Equilibrium unfolding is used to determine the conformational stability of the molecule.

In its simplest form, equilibrium unfolding assumes that the molecule may belong to only two thermodynamic states, the folded state (typically denoted N for "native" state) and the unfolded state (typically denoted U). This "all-or-none" model of protein folding was first proposed by Tim Anson in 1945, but is believed to hold only for small, single structural domains of proteins (Jackson, 1998); larger domains and multi-domain proteins often exhibit intermediate states. As usual in statistical mechanics, these states correspond to ensembles of molecular conformations, not just one conformation.

The molecule may transition between the native and unfolded states according to a simple kinetic model

with rate constants ${\displaystyle k_{f}}$ and ${\displaystyle k_{u}}$ for the folding (${\displaystyle {\ce {U->N}}}$) and unfolding (${\displaystyle {\ce {N->U}}}$) reactions, respectively. The dimensionless equilibrium constant ${\displaystyle K_{eq}\ {\stackrel {\mathrm {def} }{=}}\ {\frac {k_{u}}{k_{f}}}={\frac {\left[{\ce {U}}\right]_{eq}}{\left[{\ce {N}}\right]_{eq}}}}$ can be used to determine the conformational stability ${\displaystyle \Delta G^{o}}$ by the equation

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