Folding pathway mediated by an intramolecular chaperone.

Abstract

The N-terminal propeptide of subtilisin, a serine protease, functions as an intramolecular chaperone which is crucial for proper folding of the active enzyme. This nascent N-terminal propeptide is removed after completion of the folding process. Here we present a possible pathway by which intramolecular chaperones mediate protein folding. Using circular dichroism to analyze acid-denatured subtilisin we have identified a folding-competent state which can refold to an active conformation in the absence of the propeptide. Earlier work had shown that guanidine hydrochloridedenatured subtilisin was in a state incapable of folding in absence of its propeptide. Comparison of the foldingincompetent and folding-competent states indicates that refolding is facilitated by the presence of residual structure present only in the folding-competent state. The analysis further indicates that the propeptide is essential for inducing this state. Therefore the folding-competent state may lie on-or be in rapid equilibrium with an intermediate on-the folding pathway of subtilisin. In the absence of the propeptide, formation of such a state-and hence refolding-is extremely slow.A large number of proteases, both in prokaryotes and in eukaryotes, are synthesized as precursors with N-terminal prosegments which play a vital role in the folding pathway (1). Subsequently, these propeptides are cleaved proteolytically to generate active enzymes. Hence, proteins such as subtilisins (2, 3), a-lytic protease (4), and carboxypeptidase (5), when unfolded in guanidine hydrochloride (Gdn'HCl) solution, refold only in the presence of their propeptides. Using subtilisin E as a model system, work in our laboratory has unambiguously shown that the 77-amino acid propeptide plays a vital role in folding the 275 amino acids of the mature enzyme (6). Because the propeptide is covalently attached to the N terminus of the mature enzyme prior to its maturation, and because each propeptide molecule mediates the folding of one enzyme molecule (generally the corresponding molecule), the term intramolecular chaperones has been coined for such propeptides to distinguish them from chaperones (6-8). Concurrent with this proposition, it was shown that the 13-amino acid propeptide of bovine pancreatic trypsin inhibitor facilitated the in vitro folding pathway by acting as a tethered, solvent-accessible, intramolecular thiol-disulfide reagent (9). Recently, it was shown that the propeptide of a-lytic protease helped to overcome a kinetic block in the folding pathway (10). of subtilisin facilitates folding in a manner different from that of a-lytic protease.Earlier work in our laboratory showed that in vitro the propeptide from subtilisin E could refold not only subtilisin E but also subtilisin BPN' and subtilisin Carlsberg. All three of these subtilisins are highly homologous and have very similar three-dimensional structures. In the present work, we have taken advantage of the ability of the propeptide of subtilisin E to facilitate refolding of subtilisin B...

Irreversible thermal denaturation ofTorpedo californicaacetylcholinesterase

“…The authors suggested that the pro region permits folding to occur at a measurable rate by lowering the free energy barrier separating the denatured state from the thermodynamically more favored native state. Similar findings have been presented for carboxypeptidase Y (Smensen et al, 1993) and subtilisin (Eder et al, 1993;Shinde et al, 1993). However, AChE lacks a pro region (Maulet et al, 1990), and kinetic trapping of AChE in the MG state must be due to some other reason.…”

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF

“…[32][33][34][35] The uncleaved, contiguous propeptide functions at only one point in the folding pathway: assisting in protein folding and stabilization prior to protein maturation. 32 These chaperones remain attached to the mature sequence until the maturation process is complete, and then are proteolytically removed by autoprocessing or another endopeptidase. 32 Analogous to other intramolecular chaperones, the lengthy propeptide (741 aa) of VWF appears to stabilize the intramolecular disulfide interactions between cysteines within individual D1, D2, and D3 domains.…”

“…32 These chaperones remain attached to the mature sequence until the maturation process is complete, and then are proteolytically removed by autoprocessing or another endopeptidase. 32 Analogous to other intramolecular chaperones, the lengthy propeptide (741 aa) of VWF appears to stabilize the intramolecular disulfide interactions between cysteines within individual D1, D2, and D3 domains. It is our hypothesis that the propeptide of VWF functions during the maturation process as an intramolecular chaperone.…”

Sucrase Is an Intramolecular Chaperone Located at the C-terminal End of the Sucrase-Isomaltase Enzyme Complex

“…A growing family of membrane and secretory proteins contain pro-domains at the N-terminal end that undergo post-translational proteolytic cleavage in the late secretory pathway after the protein has acquired transport competence (26,(33)(34)(35)(36). These domains, called intramolecular chaperones, modulate the folding of this class of proteins directly through interaction with other regions of the protein or by substituting for ER resident chaperones.…”