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

Stina Lindman1, Armando Hernandez-Garcia, Olga Szczepankiewicz

  • 1Center for Molecular Protein Science, Biochemistry and Biophysical Chemistry, Lund University, SE-22100 Lund, Sweden.

Proceedings of the National Academy of Sciences of the United States of America
|November 3, 2010
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Summary
This summary is machine-generated.

This study enhanced protein stability using a split green fluorescent protein (GFP) system. Mutations improving fragment binding also increased overall protein stability, yielding a 12°C higher thermal denaturation midpoint.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Protein stability is crucial for function and therapeutic applications.
  • In vivo screening methods offer efficient ways to identify protein variants with improved properties.
  • The thermodynamic linkage between protein folding and fragment complementation can be exploited for stability engineering.

Purpose of the Study:

  • To develop a novel in vivo screening method for enhancing protein stability.
  • To identify protein variants with increased thermodynamic stability using a split green fluorescent protein (GFP) system.
  • To investigate the correlation between fragment binding affinity and overall protein stability.

Main Methods:

  • Utilized a split GFP system for in vivo screening of protein variants.
  • Generated a library of PGB1 protein fragments (1-40) and screened against wild-type fragments (41-56).
  • Assessed protein stability through thermal denaturation assays and quantified changes in midpoint temperature and free energy of stabilization.

Main Results:

  • Identified PGB1 mutants with significantly higher affinity between split GFP fragments.
  • Demonstrated a direct correlation between fragment binding affinity and enhanced overall protein stability.
  • The top mutant exhibited a 12°C increase in thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25°C.
  • Stabilization mechanisms included increased hydrophobic effect, optimized van der Waals interactions, helix stabilization, improved hydrogen bonding, and reduced electrostatic repulsion.

Conclusions:

  • The split GFP system is a viable tool for in vivo protein stabilization through fragment complementation screening.
  • Mutations enhancing fragment affinity can effectively improve overall protein stability.
  • The identified stabilization strategies provide insights into protein engineering for enhanced robustness.