The mutations in the super Spy variants altered a region on the surface of Spy, which additional experiments revealed was likely to be involved in binding to the partner proteins. All of these variants were also better than the wild-type Spy at stabilising two other unfolded proteins-and so were dubbed ‘super Spy’ proteins. This assay identified several variants of Spy that could stabilise an unstable test protein even more effectively than the wild-type Spy can. have now used it to find variants of this protein that perform as even better chaperones.

This approach was formerly used to identify a new chaperone called Spy, and Quan et al. have tackled this challenge by using a bacterial assay-that they had developed previously-and which links the correct folding of a test protein to cell survival and growth in the presence of an antibiotic. However, studying this hypothesis directly has proven to be difficult. Additional proteins called chaperones often help with this folding process, and it has been proposed that chaperones must be particularly flexible in order to cope with the changes in the shape of the different proteins being folded. Folding into the correct shape is crucial for a protein to function properly because many proteins work by binding to certain other proteins or molecules, like a key fitting into a lock. Proteins are made from long chains of smaller molecules, called amino acids, that twist and fold into complex three-dimensional shapes. Our results provide evidence for the importance of disorder and flexibility in chaperone function. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. Experimental study of the role of disorder in protein function is challenging.

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