Synthesis of proteins containing modified arginine residues.
Unnatural amino acid mutagenesis provides the wherewithal to study protein function in great detail. To extend the repertoire of functionalized amino acids available for study by this technique, seven structural analogues of arginine were prepared and used to activate a suppressor tRNACUA. These included Ngamma-methylarginine, Ngamma-nitroarginine, citrulline, homoarginine, and three conformationally constrained analogues based on proline. These misacylated tRNAs were shown to be capable of introducing the arginine analogues into dihydrofolate reductase (position 22) and Photinus pyralis luciferase (positions 218 and 437). Most of the modified luciferases containing arginine analogues at position 218 emitted light with less efficiency and at longer wavelength than the wild type. This is consistent with the postulated role of this residue as essential for maintaining the polarity and rigidity of the luciferin-binding site. Interestingly, the luciferase containing Ngamma-methylarginine at position 218 emitted light at the same wavelength as the wild type and was at least as efficient. Alteration of the arginine residue at position 437 had no effect on the wavelength of emitted light but afforded analogues, all of which emitted light less efficiently than the wild type. This is altogether consistent with the putative role of Arg437, which is an invariant residue within the superfamily of enzymes that includes P. pyralis luciferase. This amino acid is part of the linker between the two structural domains of luciferase that is believed to be essential for efficient enzyme function but not part of the substrate-binding site.
Choudhury
Ambar K
AK
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904-4319, USA..
Golovine
Serguei Y
SY
Dedkova
Larisa M
LM
Hecht
Sidney M
SM
D000595:Amino Acid Sequence; D001120:Arginine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D049409:Luciferases, Firefly / Q000235:genetics; D008958:Models, Molecular; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D012346:RNA, Transfer, Amino Acyl; C485197:luciferase, Photinus; D001120:Arginine; D049409:Luciferases, Firefly; D013762:Tetrahydrofolate Dehydrogenase