Local energetic frustration affects the dependence of green fluorescent protein folding on the chaperonin GroEL

Abstract

The GroE chaperonin system in comprises GroEL and GroES and facilitates ATP-dependent protein folding and Proteins with very similar sequences and structures can differ in their dependence on GroEL for efficient folding. One potential but unverified source for GroEL dependence is frustration, wherein not all interactions in the native state are optimized energetically, thereby potentiating slow folding and misfolding. Here, we chose enhanced green fluorescent protein as a model system and subjected it to random mutagenesis, followed by screening for variants whose folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with folding assays. Strikingly, mutations at positions predicted to be highly frustrated were found to correlate with decreased GroEL dependence. Conversely, mutations at positions with low frustration were found to correlate with increased GroEL dependence. Further support for this finding was obtained by showing that folding of an enhanced green fluorescent protein variant designed computationally to have reduced frustration is indeed less GroEL-dependent. Our results indicate that changes in local frustration also affect partitioning between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.

Mechanisms of Chaperones as Active Assistant/Protector for Proteins: Insights from NMR Studies

“…The same binding site on SH3 was also found to be engaged in interaction with the chaperonin GroEL, further supporting the notion that recognizing locally frustrated regions could be a common principle for chaperone‐client interactions where the client is a natively well‐folded protein . Moreover, a random mutagenesis study of GroEL‐client interaction using green fluorescent protein (GFP) as a model substrate demonstrated that mutations at highly frustrated positions correlate with decreased GroEL dependence during folding, whereas mutations at low frustration positions correlate with increased GroEL dependence . The results provide direct evidence for the link between protein frustration and chaperone function.…”

Influence of Fluorescent Protein Maturation on FRET Measurements in Living Cells

“… 18 We mutated a single amino acid in mTurquoise2, in an attempt to increase its maturation rate. Because the reaction with oxygen is a rate-determining step (although folding contributes as well), 27 we reasoned that if we could force the desired conformation onto the tyrosine side chain to undergo the reaction with oxygen, generating the planar alkene of the fluorophore, we would accelerate maturation. Residue 203 can interact with the tyrosine phenol, 28 , 29 and we mutated the threonine 203 to an isoleucine, which is nonpolar and would lead to a net increase in attraction with the nonpolar aromatic ring, and thus perhaps assist in the maturation reaction by directing the tyrosine.…”

Comparative genomic analysis of mollicutes with and without a chaperonin system

“…This observation is consistent with the report [ 31 ] that single amino acid changes are sufficient to convert a GroE-independent protein into a dependent one. Furthermore, in a recent study [ 32 ] we studied the GroEL dependence of GFP, a eukaryotic protein that is often used as a fluorescent marker also in prokaryotic systems and folds in a GroE-dependent manner. We found that single mutations in GFP can decrease the GroEL dependence of its folding.…”