A facile approach for incorporating tyrosine esters to probe ion-binding sites and backbone hydrogen bonds
Year: 2023
Type: article
Abstract: Amide-to-ester substitutions are used to study the role of the amide bonds of the protein backbone in protein structure, function, and folding. An amber suppressor tRNA/synthetase pair has been reported for incorporation of p-hydroxy-phenyl-L-lactic acid (HPLA), thereby introducing ester substitution at tyrosine residues. However, the application of this approach was limited due to the low yields of the modified proteins and the high cost of HPLA. Here we report the in vivo generation of HPLA from the significantly cheaper phenyl-L-lactic acid. We also construct an optimized plasmid with the HPLA suppressor tRNA/synthetase pair that provides higher yields of the modified proteins. The combination of the new plasmid and the in-situ generation of HPLA provides a facile and economical approach for introducing tyrosine ester substitutions. We demonstrate the utility of this approach by introducing tyrosine ester substitutions into the K + channel KcsA and the integral membrane enzyme GlpG. We introduce the tyrosine ester in the selectivity filter of the M96V mutant of the KcsA to probe the role of the second ion binding site in the conformation of the selectivity filter and the process of inactivation. We use tyrosine ester substitutions in GlpG to perturb backbone H-bonds to investigate the contribution of these H-bonds to membrane protein stability. We anticipate that the approach developed in this study will facilitate further investigations using tyrosine ester substitutions.
Source: Journal of Biological Chemistry
Authors: Ravikumar Reddi, Satyaki Chatterjee, Kimberly Matulef, Andrew Gustafson, Lujia Gao, Francis I. Valiyaveetil
Institution: Oregon Health & Science University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,800
Awards: G1286236842, G2539837713, G3083819904, Probing functional mechanisms in K+ channels using unnatural mutagenesis, G382641700, G419881185, G42916606, G4501827968, G498139845, G6188209572, G6206027980, G8229677252, G9000054807, G9000263589, ALS Efficiently Networking Advanced Beam Line Experiments (ALS-ENABLE), G969889393
OpenAlex ID: W4389158638