Monitoring Protein Global and Local Parameters in Unfolding and Binding Studies: The Extended Applicability of the Diffusion CoefficientMolecular Size Empirical Relations

Protein
unfolding and denaturation are main issues in biochemical
and pharmaceutical research. Using a global parameter, the translational
diffusion coefficient D, folded, unfolded, and intrinsically
disordered proteins of a given molar mass M can be
distinguished based on their distinct hydrodynamic properties. For
broader applications, we provide generalized, PFG-NMR-based empirical D–M relations validated at different temperatures
and ready to use with the corresponding corrections in different media.
We demonstrate that these relations enable a more accurate molecular
mass determination and show fewer potential errors than those of the
common methods based on small-molecular diffusion standards. We monitor
unfolding of three model proteins using 8 M urea and dimethyl sulfoxide
(DMSO)–water mixtures as denaturing agents, highlighting the
effect of disulfide bonds. Denaturation in 8 M urea is pH-dependent;
in addition, for proteins with highly stable disulfide bonds, a reducing
agent (TCEP) is required to achieve complete unfolding. Regarding
the effect of local parameters, we show that at low DMSO concentrationscommon
conditions in pharmaceutical binding studiesthe PFG-NMR-derived
global parameters are not significantly affected. Still, the atomic
environments can change, and the bound solvent molecule can inhibit
the binding of a partner molecule. Using proteins with natural isotopic
abundance, this effect can be proven by fast 1H–15N 2D correlation spectra. Our results enable fast and easy
estimation of protein molecular mass and the degree of folding in
various media; moreover, the effect of the cosolvent on the atomic-level
structure can be traced without the need of isotope labeling.