Abstract
Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins containing new building blocks with unique structures, chemistry, and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb critical function of a target protein. This study investigates how the site-specific incorporation of NNAAs affects catalytic properties of an enzyme. A NNAA with a hydrophobic and bulky sidechain, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at six different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals volume upon 2Nal incorporation exhibited a greater reduction in the catalytic efficiency. Similarly, the steric incompatibility calculated using RosettaDesign, a protein stability calculation program, correlated with the changes in the catalytic efficiency.
Highlights
Protein engineering frequently creates useful proteins with improved or new functions
Since solvent accessibility is highly restricted within this area, we restricted our selection to residues (PDB ID: 2W3M) with an accessible solvent area (ASA) less than or equal to 2.0% [44,60]
The two conservative mutations W113Z and F134Z led to the modest reduction in catalytic efficiency, while non-conservative mutations V50Z, I51Z, V112Z, and V135Z reduced the catalytic efficiency into a level less than 12% of that of mDHFRWT-yPheRSnaph encoding wild-type DHFR (mDHFRWT)
Summary
Protein engineering frequently creates useful proteins with improved or new functions. The study focused primarily on the effect of site-specific incorporation of NNAAs into the hydrophobic core region, where a mutation can significantly perturb the local and/or global structure of the enzyme. Since the structure of the hydrophobic core can be governed by its conformational stability, six NNAA incorporation sites were selected from the hydrophobic core of the mDHFR in order to investigate how incorporation of a hydrophobic non-natural amino acid affects the conformational stability and catalytic function of mDHFR. While the lower size spectrum of hydrophobic residues is well covered by the natural amino acids in nature, the use of larger sidechains is limited This presented an opportunity to investigate whether larger hydrophobic amino acids could be successfully incorporated into the enzyme core region, while maintaining enzyme function. Since 2Nal is bulkier than any natural amino acids, incorporation of 2Nal into the hydrophobic core of mDHFR was expected to significantly perturb the structure and stability of the mDHFR
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
