Verification of the Stabilized Protein Design Based on the Prediction of Intrinsically Disordered Regions: Ribosomal...

Area of Science:

• Protein Engineering
• Structural Biology
• Bioinformatics

Background:

• Intrinsically disordered region (IDR) prediction programs can identify weakened sites in protein sequences.
• These predicted sites are potential targets for introducing protein-stabilizing mutations.
• The relative importance of amino acid sequence versus 3D structure in determining protein stabilization remains unclear.

Purpose of the Study:

• To investigate whether protein sequence (and predicted weakened sites) or 3D structure is the primary determinant of protein stabilization.
• To compare the effects of identical mutations in proteins with similar structures but different sequences.

Main Methods:

• Selected two ribosomal protein L1 variants: Haloarcula marismortui L1 (HmaL1) and Aquifex aeolicus L1 (AaeL1), which share similar 3D structures but differ in amino acid sequence.
• Introduced identical disulfide bond mutations into regions predicted differently for sequence-based stability.
• Measured the impact of mutations on protein melting temperature (Tm) as an indicator of stability.

Main Results:

• A disulfide bond in a predicted structured region of AaeL1 did not increase its melting temperature.
• A disulfide bond in the corresponding region of HmaL1, predicted as weakened, increased the melting temperature by approximately 10°C.
• This suggests sequence-based predictions of weakened sites are crucial for successful stabilization.

Conclusions:

• Protein stabilization is significantly influenced by targeting predicted weakened sites in the amino acid sequence.
• Sequence-based predictions are more critical for guiding protein stabilization strategies than solely relying on conserved 3D structures.
• This finding has implications for protein engineering and rational design of more stable proteins.