Structural Events during the Refolding of an All β-Sheet Protein

Abstract

The refolding kinetics of the 140-residue, all ␤-sheet, human fibroblast growth factor (hFGF-1) is studied using a variety of biophysical techniques such as stoppedflow fluorescence, stopped-flow circular dichroism, and quenched-flow hydrogen exchange in conjunction with multidimensional NMR spectroscopy. Urea-induced unfolding of hFGF-1 under equilibrium conditions reveals that the protein folds via a two-state (native 7 unfolded) mechanism without the accumulation of stable intermediates. However, measurement of the unfolding and refolding rates in various concentrations of urea shows that the refolding of hFGF-1 proceeds through accumulation of kinetic intermediates. Results of the quenchedflow hydrogen exchange experiments reveal that the hydrogen bonds linking the N-and C-terminal ends are the first to form during the refolding of hFGF-1. The basic ␤-trefoil framework is provided by the simultaneous formation of ␤-strands I, IV, IX, and X. The other ␤-strands comprising the ␤-barrel structure of hFGF-1 are formed relatively slowly with time constants ranging from 4 to 13 s.

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

“…Finally, refinement of anisotropic displacement parameters from a 1.10 Å X-ray diffraction dataset suggests that the N-and C-terminal regions of FGF-1 do not form a rigid body; instead, the N-and C-termini appear to be sliding past one another, suggestive of a tenuous interaction (even when stabilized by the folded structure). 51 Thus, our analysis of the folding of FGF-1 is inconsistent with the published report of Yu and coworkers, 48 but is in general agreement with the folding studies of other b-trefoil proteins in which the central strands of the protein are observed to fold faster than those on the periphery. 47,52,53 Only a key subset of structural elements (turns #2-#5 and turn #7, spanning $50% of the overall protein) appears necessary to confer efficient foldability to FGF-1.…”

“…The basis for this conclusion was a report that probed the folding of FGF-1 using NMR H/D exchange studies and identified the association of the N-and C-termini as the first step in the folding pathway. 48 The u-value analysis presented here is inconsistent with this proposed folding pathway: both turns #1 and #11 are observed to fold after barrier crossing, and neither turn is part of the folding nucleus (which approximately spans turns #2-#7). In the absence of significant residual structure, termini closure as the first step in folding seems unlikely from an entropic standpoint, as it would require association of the two most distal structural elements of the protein.…”

Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

“…It has been shown that the folding/unfolding of small globular proteins occurs via a two-state process, whereas the folding/ unfolding of larger proteins (Ͼ100 amino acids) is complex and often involves the formation of intermediate(s) (1)(2)(3). The most thorough investigations of protein folding and stability have been done with unusually small proteins, which are folded into single domains and display simple two-state unfolding processes.…”

Identification of Rare Partially Unfolded States in Equilibrium with the Native Conformation in an All β-Barrel Protein

“…2). We recently investigated the chronology of events in the refolding pathway of hFGF-1 from its urea-unfolded state(s) (50). The Chevron plot (natural logarithm of the unfolding/refolding rate constant versus the denaturant concentration) shows a prominent curvature at low concentrations of urea (Fig.…”