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The Hsp90 cycle depends on the coordinated activity of a range of cochaperones, including Hop, Hsp70 and peptidyl-prolyl isomerases such as FKBP52. Using mass spectrometry, we investigate the order of addition of these cochaperones and their effects on the stoichiometry and composition of the resulting Hsp90-containing complexes. Our results show that monomeric Hop binds specifically to the Hsp90 dimer whereas FKBP52 binds to both monomeric and dimeric forms of Hsp90. By preforming Hsp90 complexes with either Hop, followed by addition of FKBP52, or with FKBP52 and subsequent addition of Hop, we monitor the formation of a predominant asymmetric ternary complex containing both cochaperones. This asymmetric complex is subsequently able to interact with the chaperone Hsp70 to form quaternary complexes containing all four proteins. Monitoring the population of these complexes during their formation and at equilibrium allows us to model the complex formation and to extract 14 different KD values. This simultaneous calculation of the KDs from a complex system with the same method, from eight deferent datasets under the same buffer conditions delivers a self-consistent set of values. In this case, the KD values afford insights into the assembly of ten Hsp90-containing complexes and provide a rationale for the cellular heterogeneity and prevalence of intermediates in the Hsp90 chaperone cycle. [ABSTRACT FROM AUTHOR]

Structures that contain a knot formed by the path of the polypeptide backbone represent some of the most complex topologies observed in proteins. How or why these topological knots arise remains unclear. By developing a method to experimentally trap and detect knots in nonnative polypeptide chains, we find that two knotted methyltransferases, YibK and YbeA, can exist in a trefoil-knot conformation even in their chemically unfolded states. The unique denatured-state topology of these molecules explains their ability to efficiently fold to their native knotted structures in vitro and offers insights into the potential role of knots in proteins. Furthermore, the high prevalence of the denatured-state knots identified here suggests that they are either difficult to untie or that threading of any untied molecules is rapid and spontaneous. The occurrence of such knotted topologies in unfolded polypeptide chains raises the possibility that they could play an important, and as yet unexplored, role in folding and misfolding processes in vivo. [ABSTRACT FROM AUTHOR]

The tetratricopeptide repeat (TPR) is a 34-aa a-helical motif that occurs in tandem arrays in a variety of different proteins. In natural proteins, the number of TPR motifs ranges from 3 to 16 or more. These arrays function as molecular scaffolds and frequently mediate protein-protein interactions. We have shown that correctly folded TPR domain proteins, exhibiting the typical helix-turn-helix fold, can be designed by arraying tandem repeats of an idealized TPR consensus motif. To date, three designed proteins, CTPR1, CTPR2, and CTPR3 (consensus TPR number of repeats) have been characterized. Their high-resolution crystal structures show that the designed proteins indeed adopt the typical TPR fold, which is specified by the correct positioning of key residues. Here, we present a study of the thermodynamic properties and folding kinetics of this set of designed proteins. Chemical denaturation, monitored by CD and fluorescence, was used to assess the folding and global stability of each protein. NMR-detected amide proton exchange was used to investigate the stability of each construct at a residue-specific level. The results of these studies reveal a stable core, which defines the intrinsic stability of an individual TPR motif. The results also show the relationship between the number of tandem repeats and the overall stability and folding of the protein. [ABSTRACT FROM AUTHOR]