8DHR
An ester mutant of SfGFP
Summary for 8DHR
| Entry DOI | 10.2210/pdb8dhr/pdb |
| Descriptor | Green fluorescent protein (2 entities in total) |
| Functional Keywords | beta barrel, ester mutant, fluorescent protein |
| Biological source | Aequorea victoria |
| Total number of polymer chains | 2 |
| Total formula weight | 55546.39 |
| Authors | |
| Primary citation | Reddi, R.,Chatterjee, S.,Matulef, K.,Gustafson, A.,Gao, L.,Valiyaveetil, F.I. A facile approach for incorporating tyrosine esters to probe ion-binding sites and backbone hydrogen bonds. J.Biol.Chem., 300:105517-105517, 2023 Cited by PubMed 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. PubMed: 38042487DOI: 10.1016/j.jbc.2023.105517 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.75 Å) |
Structure validation
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