Persistence of Native-Like Topology in a Denatured Protein in 8 M Urea

Abstract

Experimental methods have demonstrated that when a protein unfolds, not all of its structure is lost. Here we report measurement of residual dipolar couplings in denatured forms of the small protein staphylococcal nuclease oriented in strained polyacrylamide gels. A highly significant correlation among the dipolar couplings for individual residues suggests that a native-like spatial positioning and orientation of chain segments (topology) persists to concentrations of at least 8 molar urea. These data demonstrate that long-range ordering can occur well before a folding protein attains a compact conformation, a conclusion not anticipated by any of the standard models of protein folding.

Protein folding: Then and now

“…Under denaturing conditions, the protein can still exhibit native conformational bias and retain a certain amount of residual native structures. Mounting experimental evidence [168,[195][196][197][198] supports residual native-like structural elements in the denatured state for a variety of proteins. Using residual dipolar coupling from NMR measurements, Shortle and Ackerman [195] showed that native-like topology persists under strong denaturing conditions as high as 8 M Urea for a truncated staphylococcal nuclease.…”

“…Mounting experimental evidence [168,[195][196][197][198] supports residual native-like structural elements in the denatured state for a variety of proteins. Using residual dipolar coupling from NMR measurements, Shortle and Ackerman [195] showed that native-like topology persists under strong denaturing conditions as high as 8 M Urea for a truncated staphylococcal nuclease. By applying quasi-elastic neutronscattering on α-lactalbumin, Bu et al [196] demonstrated residual helical structure and tertiarylike interactions even in the absence of disulfide bonds and under highly denaturing conditions.…”

“…Unfolded proteins structure Unfolded proteins' NMR [195]. Size of measurements of various unfolded proteins [192].…”

Kinetic stability of membrane proteins

“…The reversible denatured state of soluble proteins has been characterized as an ensemble of multiple conformations with a very low content of secondary and tertiary structure (Shortle and Ackerman 2001). Contrasting with the observed parallelism in the unfolding energetics between membrane and soluble proteins, helical membrane proteins seem to retain significant secondary structure in the unfolded states Roman et al 2010).…”

Theory of Phase Transitions in Polypeptides and Proteins

“…states [167]. Thus, while the rigid native-like core of the protein is formed, a significant fraction of amino acids in the flexible regions of the protein can exist in the unfolded state.…”