ArticleProceedings of the National Academy of Sciences of the United States of America2010
In vivo protein stabilization based on fragment complementation and a split GFP system.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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14 citing papers in PubMed, 30 citations in OpenAlex.
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- An enzyme-based biosensor for monitoring and engineering protein stability in vivo.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- In Vitro Assays: Friends or Foes of Cell-Penetrating Peptides.International journal of molecular sciences · 2020Review
- Split Green Fluorescent Proteins: Scope, Limitations, and Outlook.Annual review of biophysics · 2019Review
- Protein stabilization with retained function of monellin using a split GFP system.Scientific reports · 2018Article
- A putative RNA binding protein fromFEBS open bio · 2018Article
- Directed evolution to improve protein folding in vivo.Current opinion in structural biology · 2018Review
- Hysteresis as a Marker for Complex, Overlapping Landscapes in Proteins.The journal of physical chemistry letters · 2013Article
- Comprehensive analysis of LANA interacting proteins essential for viral genome tethering and persistence.PloS one · 2013Article
- Split-protein systems: beyond binary protein-protein interactions.Current opinion in chemical biology · 2011Review
- Protein stability by number: high-throughput and statistical approaches to one of protein science's most difficult problems.Current opinion in chemical biology · 2011Review
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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