IPRO+/-: Computational Protein Design Tool Allowing for Insertions and Deletions

Area of Science:

• Protein engineering
• Computational biology
• Enzyme design

Background:

• Insertions and deletions (indels) in proteins alter residue spacing, offering unique functional tuning beyond substitutions.
• Computational protein design traditionally focuses on amino acid substitutions, limiting exploration of indel effects.

Purpose of the Study:

• To develop an optimization-based computational approach for protein redesign incorporating indels and substitutions.
• To predict beneficial indel and substitution combinations for novel protein variants.
• To provide tools for enzyme engineering and broader protein design tasks.

Main Methods:

• Developed an optimization-based computational protein redesign algorithm.
• Integrated prediction of beneficial indels and substitutions.
• Generated putative substrate-docked structures for designed protein variants.
• Utilized PyRosetta to create the open-source Indel-Maker tool.

Main Results:

• Identified active variants of bacterial thioesterase ('TesA) with experimental validation.
• Successfully recapitulated known active TEM-1 β-Lactamase sequences of varying lengths.
• Discovered shorter 4-Coumarate:CoA ligase variants with enhanced in vitro activity on non-native substrates.

Conclusions:

• Computational indel incorporation enables novel protein engineering strategies.
• The developed method and Indel-Maker tool facilitate the design of enzymes with tailored functions and sizes.
• This approach expands the possibilities for tuning protein function through sequence manipulation.