Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu→Glu−) to destabilize and unfold a specific region of the protein. We apply this strategy to Ubiquitin, reversibly trapping a folding intermediate in which the β5 strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high energy states.

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β (Aβ), peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two Cterminal glycines (k cat = 2 sec -1 ) followed by a slow cleavage between residues 72-73 (k cat = 0.07 sec -1 ), thereby producing the inactive Ub1-74 and Ub1-72. IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼90 fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG ‹ 0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE. Accession Number: The atomic coordinates and structure factors (PDB ID: 3OFI) have been deposited in the Protein Data Bank,

Insulin‐degrading enzyme (IDE) is a zinc metalloprotease involved in the clearance of small bioactive peptides, such as insulin and amyloid‐beta. Given the considerable diversity in substrate tertiary structure, the criteria for substrate recognition and degradation by this protease have been difficult to determine. Here, we evaluate size and local stability as parameters in the substrate selectivity of IDE using opioid peptides and ubiquitin variants as model systems, respectively. Kinetic analyses of IDE, using opioid peptides ranging from 5 to 31 residues, demonstrate that opioid peptides containing 17 or more residues exhibit Km values of 50 μM or less and kcat values that do not exceed 40 s−1. In contrast, smaller opioid peptides exhibited Km values above 900 μM and kcat values above 600 s−1. Mutations at conserved substrate binding sites in IDE are currently being investigated to identify the molecular basis of substrate size discrimination. To probe local stability as a parameter in substrate selectivity, we mutated residues on each of the seven major structural elements of ubiquitin (Ub), a 76‐residue inhibitor of IDE, which destabilize Ub by 0.5–9 kcal/mol. Surprisingly, Ub variants which lose at least 1.6 kcal/mol in local stability are cleaved by IDE with kcat values above 0.5 sec−1. While the first cut site is identical in all digested variants, the cleavage pattern varies with increasing instability of Ub proteins. Together, these findings imply that changing a substrate's property modifies the enzyme's specificity and thus suggest a knowledge basis for the rational design of peptidomimetic modulators of IDE activity.