Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease

Area of Science:

• Biochemistry
• Organic Chemistry
• Molecular Biology

Background:

• The chemical synthesis of proteins, particularly enzymes like HIV-1 protease, faces challenges with peptide segment solubility.
• The active site flaps of HIV-1 protease are sensitive to amino acid mutations, impacting enzyme function.

Purpose of the Study:

• To develop a novel total chemical synthesis approach for preparing backbone-engineered HIV-1 protease.
• To create a fully active HIV-1 protease analog with modified peptide bonds in the active site flaps.

Main Methods:

• Utilized a total chemical synthesis strategy involving the ligation of unprotected synthetic peptide segments.
• Employed mutually reactive functional groups for clean and high-yield peptide joining.
• Performed ligation in 6 M guanidine hydrochloride to address solubility limitations.

Main Results:

• Successfully synthesized a backbone-engineered HIV-1 protease analog with a thioester linkage replacing the natural peptide bond between Gly51-Gly52 in each flap.
• The synthesized analog demonstrated full enzymatic activity.
• The method circumvented the solubility issues associated with protected peptide segments in classical protein synthesis.

Conclusions:

• A robust total chemical synthesis method was established for producing backbone-engineered HIV-1 protease analogs.
• The modified HIV-1 protease analog retains full activity, indicating the potential for such modifications in enzyme engineering.
• This approach offers a viable alternative for synthesizing complex proteins and their analogs.