Conformational Bias in Unfolded Proteins Studied by Sequence Reversal

Abstract

Sequence patterns of charge, hydrophobicity, hydrogen bonding, and other amino acid physicochemical properties contribute to mechanisms of protein folding, but how sequence composition and patterns influence the conformational dynamics of the denatured state ensemble is not fully understood. To investigate structure-sequence relationships in the denatured state, we reversed the sequence of staphylococcal nuclease and characterized its structure, thermodynamic character, and hydrodynamic radius using circular dichroism spectroscopy, dynamic light scattering, analytical ultracentrifugation, and size-exclusion chromatography as a function of temperature. The macromolecular size of “Retro-nuclease” was found to be highly expanded in solution with characteristics similar to biological intrinsically disordered proteins. In contradistinction to a disordered state, Retro-nuclease exhibits a broad sigmoid transition of its hydrodynamic dimensions as temperature is increased, indicating a thermodynamically controlled compaction. Counterintuitively, the magnitude of these temperature-induced hydrodynamic changes exceed that observed from thermal denaturation of folded unaltered staphylococcal nuclease. Undetectable by calorimetry and intrinsic tryptophan fluorescence, the lack of heat capacity or fluorescence changes throughout the thermal transition indicate canonical hydrophobic collapse did not drive the Retro-nuclease structural transitions. Instead, temperature-dependent circular dichroism spectroscopy and computer simulation correlate the temperature sensitivity to the intrinsic sampling of backbone conformations for polyproline II and α-helix. Using analytical techniques that relate backbone conformational bias to hydrodynamic size, the observed changes in Retro-nuclease structure with temperature indicate backbone conformational propensities and coil transition enthalpies that are in good agreement with calorimetric and spectroscopic studies of short peptides.