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

Boudhayan Bandyopadhyay, Adi Goldenzweig, Tamar Unger, Orit Adato, Sarel J. Fleishman, Ron Unger, Amnon Horovitz

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


The GroE chaperonin system in Escherichia coli comprises GroEL and GroES and facilitates ATP-dependent protein foldingin vivo and in vitro. 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 in vivo folding displays increased or decreased GroEL dependence. We confirmed the altered GroEL dependence of these variants with in vitro 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 frustrationwere foundtocorrelate with increased GroEL dependence. Further support for this finding was obtained byshowing 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 in vivo between spontaneous and chaperonin-mediated folding. Hence, the design of minimally frustrated sequences can reduce chaperonin dependence and improve protein expression levels.

Original languageEnglish
Pages (from-to)20583-20591
Number of pages9
JournalJournal of Biological Chemistry
Issue number50
StatePublished - 15 Dec 2017
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the Minerva Foundation with funding from the Federal German Ministry for Education and Research (to A. H.) and Grant 772/13 from the Israel Science Foundation (to R. U.). Research in the Fleish-man laboratory was supported by a European Research Council’s Starter’s Grant and by a donation from Sam Switzer and family. The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Tables S1 and S2 and Figs. S1 and S2. 1 Marth S. Sagon Career Development Chair. 2To whom correspondence may be addressed. E-mail: ron@biomodel. os.biu.ac.il. 3Carl and Dorothy Bennett Professorial Chair in Biochemistry. To whom corre-spondence may be addressed. E-mail: Amnon.Horovitz@weizmann.ac.il.

Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A..


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