Slow Conformational Exchange between Partially Folded\nand Near-Native States of Ubiquitin: Evidence for a Multistate Folding\nModel

The mechanism by which small proteins\nfold, i.e., via intermediates\nor via a two-state mechanism, is a subject of intense investigation.\nIntermediate states in the folding pathways of these proteins are\nsparsely populated due to transient lifetimes under normal conditions\nrendering them transparent to a majority of the biophysical methods\nemployed for structural, thermodynamic, and kinetic characterization,\nwhich attributes are essential for understanding the cooperative\nfolding/unfolding of such proteins. Dynamic NMR spectroscopy has enabled\nthe characterization of folding intermediates of ubiquitin that exist\nin equilibrium under conditions of low pH and denaturants. At low\npH, an unlocked state defined as N′ is in fast exchange with\nan invisible state, U″, as observed by CEST NMR. Addition of\nurea to ubiquitin at pH 2 creates two new states F<i>′</i> and U<i>′</i>, which are in slow exchange (<i>k</i><sub>F′→U′</sub> = 0.14 and <i>k</i><sub>U′→F′</sub> = 0.28 s<sup>–1</sup>) as indicated by longitudinal ZZ-magnetization exchange spectroscopy.\nHigh-resolution solution NMR structures of F<i>′</i> show it to be in an “unlocked” conformation with measurable\nchanges in rotational diffusion, translational diffusion, and rotational\ncorrelational times. U<i>′</i> is characterized by\nthe presence of just the highly conserved N-terminal β1−β2\nhairpin. The folding of ubiquitin is cooperative and is nucleated\nby the formation of an N-terminal β-hairpin followed by significant\nhydrophobic collapse of the protein core resulting in the formation\nof bulk of the secondary structural elements stabilized by extensive\ntertiary contacts. U<i>′</i> and F<i>′</i> may thus be described as early and late folding intermediates in\nthe ubiquitin folding pathway.