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Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability
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Folding coupled with assembly in split green fluorescent proteins studied by structure-based molecular simulations.

Mashiho Ito1, Takeaki Ozawa, Shoji Takada

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Split green fluorescent protein (GFP) folding mechanisms were simulated. Faster folding and N-terminal fragment stability correlate with successful protein-protein interaction imaging in cells.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Cell Imaging

Background:

  • Split green fluorescent protein (GFP) is vital for visualizing protein-protein interactions in living cells.
  • The folding and assembly mechanisms of split GFPs remain poorly understood.
  • Understanding these mechanisms is crucial for optimizing split GFP applications.

Purpose of the Study:

  • To investigate the molecular mechanisms governing the folding and assembly of split GFPs.
  • To correlate in silico folding dynamics with experimental observations of fluorescence recovery.
  • To elucidate the role of individual fragment folding in the overall assembly process.

Main Methods:

  • Utilized a simplified Go model based on energy landscape theory.
  • Performed comprehensive folding simulations for six split GFP variants with varying split sites.
  • Analyzed folding rates, fragment folding order, and standalone N-terminal fragment stability.

Main Results:

  • Simulations revealed that the N-terminal fragment consistently folded before the C-terminal fragment during successful assembly.
  • Higher in silico folding rates were observed for split GFPs previously reported to exhibit fluorescence recovery.
  • The stability of standalone N-terminal fragments showed a strong correlation with the overall folding rates of the split GFPs.

Conclusions:

  • Efficient folding and assembly of split GFPs depend on the N-terminal fragment folding first, using the central alpha-helix as a nucleation core.
  • C-terminal fragment folding is coupled to its assembly with the pre-folded N-terminal fragment.
  • These findings provide mechanistic insights into split GFP function and guide the design of improved variants for protein interaction studies.