V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein

Area of Science:

• Protein Science
• Biotechnology
• Structural Biology

Background:

• Inteins facilitate protein splicing, a process with significant applications in biotechnology.
• The V67L mutation in Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh) enhances stability and activity.
• Structural studies show minimal rearrangement, leaving the mechanism of V67L enhancement unclear.

Purpose of the Study:

• To elucidate the structural mechanisms behind the V67L mutation's stabilization and activation of the ΔΔIhh intein.
• To investigate how the V67L substitution impacts intein fold stability and protein splicing efficiency.

Main Methods:

• Intrinsic tryptophan fluorescence for denaturation studies.
• High-pressure nuclear magnetic resonance (NMR) to assess pressure-induced unfolding.
• Molecular dynamics (MD) simulations to analyze structural and dynamic changes.

Main Results:

• Guanidine hydrochloride denaturation revealed higher stability for ΔΔIhh-L67 (ΔGf° = -6.9 kcal mol⁻¹) compared to ΔΔIhh-V67 (ΔGf° = -4.4 kcal mol⁻¹).
• High-pressure NMR indicated ΔΔIhh-L67 is more resistant to unfolding, with a smaller folding volume change (ΔVf = 58 ± 3 mL mol⁻¹) than V67 (71 ± 2 mL mol⁻¹).
• MD simulations showed V67L reduces active site dynamics and conformational entropy, suggesting allosteric effects.

Conclusions:

• The V67L mutation enhances intein stability by filling a hydrophobic cavity, as evidenced by the difference in folding volume.
• Reduced side chain dynamics and conformational entropy at the active site contribute to increased intein activity.
• The findings provide a structural basis for understanding intein engineering for improved protein splicing applications.