Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

Area of Science:

• Biochemistry
• Structural Biology
• Membrane Protein Engineering

Background:

• Split inteins facilitate protein engineering via trans-splicing.
• Membrane proteins present unique challenges for engineering and assembly studies.

Purpose of the Study:

• To demonstrate the feasibility of split intein-mediated protein trans-splicing for membrane proteins under native conditions.
• To investigate the structural and functional integrity of a trans-spliced membrane protein.
• To explore the impact of modifications on protein function.

Main Methods:

• Dual expression and in vivo ligation of proteorhodopsin fragments.
• Circular dichroism (CD) and optical spectroscopy for fold, stability, and photodynamics.
• Solid-state NMR with isotopic labeling for in-lipid bilayer structure determination.

Main Results:

• Successfully assembled functional heptahelical proteorhodopsin from two fragments using split inteins.
• Confirmed structural and functional identity of the ligated product compared to the non-ligated protein.
• Demonstrated that BC loop modifications affect photocycle kinetics.

Conclusions:

• Protein trans-splicing is a viable method for assembling complex membrane proteins in vivo.
• The ligated proteorhodopsin is correctly folded and functionally intact.
• This approach enables novel labeling strategies and aids understanding of membrane protein assembly and interactions.