TY - JOUR
T1 - In vitro Evolution of Uracil Glycosylase Towards DnaKJ and GroEL Binding Evolves Different Misfolded States
AU - Melanker, Oran
AU - Goloubinoff, Pierre
AU - Schreiber, Gideon
N1 - Publisher Copyright:
© 2022 Elsevier Ltd
PY - 2022/7/15
Y1 - 2022/7/15
N2 - Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random mutant library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK + DnaJ + ATP (DnaKJ) or E. coli cell extract (CE), using binding to the eUNG inhibitor Ugi as probe for native fold. The CE selected population was further divided to Ugi binders (+U) or non-binders (−U). The aim here was to evaluate the sequence space and physical state of the evolved protein binding the different baits. We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection had its own trajectory, with GroEL and CE-U selecting mutants highly sensitive to protease cleavage while DnaKJ selected partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. More general, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds misfolding-prone mutant species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL- and DnaKJ-selected populations, while harboring also unique properties that can be explained by the presence of additional chaperones in CE, such as Trigger factor, HtpG and ClpB.
AB - Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random mutant library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK + DnaJ + ATP (DnaKJ) or E. coli cell extract (CE), using binding to the eUNG inhibitor Ugi as probe for native fold. The CE selected population was further divided to Ugi binders (+U) or non-binders (−U). The aim here was to evaluate the sequence space and physical state of the evolved protein binding the different baits. We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection had its own trajectory, with GroEL and CE-U selecting mutants highly sensitive to protease cleavage while DnaKJ selected partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. More general, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds misfolding-prone mutant species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL- and DnaKJ-selected populations, while harboring also unique properties that can be explained by the presence of additional chaperones in CE, such as Trigger factor, HtpG and ClpB.
KW - chaperones
KW - promiscuous binding
KW - protein-evolution
KW - protein-protein interaction
UR - http://www.scopus.com/inward/record.url?scp=85131358668&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2022.167627
DO - 10.1016/j.jmb.2022.167627
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
C2 - 35597550
AN - SCOPUS:85131358668
SN - 0022-2836
VL - 434
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 13
M1 - 167627
ER -