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In vivo protein stabilization based on fragment complementation and a split GFP system.

Lindman, Stina LU ; Hernández Garcia, Armando LU ; Szczepankiewicz, Olga LU ; Frohm, Birgitta LU and Linse, Sara LU (2010) In Proceedings of the National Academy of Sciences 107(46). p.19826-19831
Abstract
Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the... (More)
Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Proceedings of the National Academy of Sciences
volume
107
issue
46
pages
19826 - 19831
publisher
National Acad Sciences
external identifiers
  • wos:000284261800043
  • pmid:21041669
  • scopus:78650530108
ISSN
1091-6490
DOI
10.1073/pnas.1005689107
language
English
LU publication?
yes
id
f5e5a2c7-7a51-4e47-abbe-fed8b479c67c (old id 1732441)
date added to LUP
2010-12-16 14:01:28
date last changed
2018-05-29 10:17:02
@article{f5e5a2c7-7a51-4e47-abbe-fed8b479c67c,
  abstract     = {Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.},
  author       = {Lindman, Stina and Hernández Garcia, Armando and Szczepankiewicz, Olga and Frohm, Birgitta and Linse, Sara},
  issn         = {1091-6490},
  language     = {eng},
  number       = {46},
  pages        = {19826--19831},
  publisher    = {National Acad Sciences},
  series       = {Proceedings of the National Academy of Sciences},
  title        = {In vivo protein stabilization based on fragment complementation and a split GFP system.},
  url          = {http://dx.doi.org/10.1073/pnas.1005689107},
  volume       = {107},
  year         = {2010},
}