von Hippel-Lindau (VHL) disease is caused by loss of function of the VHL tumor suppressor protein. Here, we demonstrate that the folding and assembly of VHL into a complex with its partner proteins, elongin B and elongin C (herein, elongin BC), is directly mediated by the chaperonin TRiC/CCT. Association of VHL with TRiC is required for formation of the VHL-elongin BC complex. A 55-amino acid domain of VHL is both necessary and sufficient for binding to TRiC. Importantly, mutation or deletion of this domain is associated with VHL disease. We identified two mutations that disrupt the normal interaction with TRiC and impair VHL folding. Our results define a novel role for TRiC in mediating oligomerization and suggest that inactivating mutations can impair polypeptide function by interfering with chaperone-mediated folding.

The essential GroEL proteins represent a subset of molecular chaperones ubiquitously distributed among species of the eubacterial lineage, as well as in eukaryote organelles. We employed these highly conserved proteins to infer eubacterial phylogenies. GroEL from the species analyzed clustered in distinct groups in evolutionary trees drawn by either the distance or the parsimony method, which were in general agreement with those found by 16s rRNA comparisons (i.e., proteobacteria, chlamydiae, bacteroids, spirochetes, firmicutes [gram-positive bacteria], and cyanobacteria-chloroplasts). Moreover, the analysis indicated specific relationships between some of the aforementioned groups which appeared not to be clearly defined or controversial in rRNA-based phylogenetic studies. For instance, a monophyletic origin for the low-G+C and high-G+C subgroups among the firmicutes, as well as their specific relationship to the cyanobacteria-chloroplasts, was inferred. The general observations suggest that GroEL proteins provide valuable evolutionary tools for defining evolutionary relationships among the eubacterial lineage of life.

The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B. licheniformis exo small -lactamase (ES-L) have been measured. ES-L lacking 19 residues (ES-L C∆19 ) has no enzymic activity. Deletion of the last 14 residues produces ES-L C∆14 , which is 0.1% active. The enzyme lacking nine residues (ES-L C∆9 ) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme. Although ES-L C∆9 folds properly, it does so 4 orders of magnitude slower than ES-L, making possible the isolation and characterization of a compact intermediate state (I P ES-L C∆9 ). Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between I P ES-L C∆9 and other intermediate slow folding. Residues removed in ES-L C∆9 and ES-L C∆14 are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence. The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level. The results are compared with other examples of this kind in the folding literature.Although amino acid sequence determines protein conformation (1), the correspondence between sequence and structure is not exact. Some amino acid replacements are well tolerated, whereas others are disruptive (2-5). Also, we have observed that deletion of the last three residues of fatty-acid-binding protein (IFABP 1 ) causes the protein to fold into a compact state lacking tertiary structure (6), whereas an internal deletion of 17 residues fails to interfere with the formation of native IFABP (7). These considerations led us to propose that the conformational information content varies widely along the polypeptide chain and that the formation of compact states and the fine adjustment of tertiary interactions can be uncoupled by specific residue deletion (6). However, in the case of truncated IFABP, a small population of native state molecules might have escaped detection by the optical and hydrodynamic probes utilized. To avoid this obstacle, and assuming that catalytic activity is the best criterion of native structure, we now have chosen to test the proposal with exo small -lactamase (ES-L), whose biological activity, unlike that of IFABP, can be determined readily and unambiguously even at trace levels.The three-dimensional (3D) structure of ES-L is known to high-resolution (see PDB entries 2BLM and 4BLM) (8,9). This 265-residue protein has no cysteine, and it is organized in two noncontinuous domains (Figure 1): the R + domain is formed by a central, five-stranded sheet covered by three superficial R-helices; the other domain is a globular array of helical elements. The interface between the two domains harbors the catalytic site.