Biopolym. Cell. 2022; 38(1):9-16.
Structure and Function of Biopolymers
Intrinsic fluorescence of single-tryptophan form of tyrosyl-tRNA synthetase catalytic module with the replacements of Trp 87 and Trp 283 by alanine
1Blaschak I. O., 1Zayets V. N., 1Kolomiets L. A., 1Kornelyuk A. I.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143

Abstract

Aim. Mammalian tyrosyl tRNA synthetase (TyrRS) is composed of N-terminal catalytic miniTyrRS and non-catalytic C-terminal domain. After cleavage both domains of TyrRS reveal non-canonical cytokine functions. It is important to study the conformational changes of miniTyrRS in the course of ligands binding in different nanocomposite complexes. Fluorescence spectroscopy is a very powerful method to detect the local conformational changes of proteins. The study of single-tryptophan form of the protein can provide important information about flexibility and local conformational changes of the protein functional sites. Methods. Site-directed mutagenesis, bacterial expression, fluorescence spectroscopy. Results. Intrinsic fluorescence characteristics of single-tryptophan Trp40-mini TyrRS were measured, a spectral maximum at 332 nm was revealed, which corresponds to the buried state of Trp40 fluorophore in protein globule. Fluorescence quenching of Trp40 by acrylamide revealed the existence of conformational flexibility of mini TyrRS. Conclusions. Fluorescence studies of the single-tryptophan form of tyrosyl-tRNA synthetase revealed a buried state of Trp40 fluorophore but high conformational flexibility of the enzyme at the nanosecond time scale.
Keywords: tyrosyl-tRNA synthetase, fluorescence spectroscopy, mutant form of miniTyrRS, active site, conformational flexibility

References

[1] Mirande M. Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: structural domains and their implications. Prog Nucleic Acid Res Mol Biol. 1991;40:95-142.
[2] Pang YL, Poruri K, Martinis SA. tRNA synthetase: tRNA aminoacylation and beyond. Wiley Interdiscip Rev RNA. 2014;5(4):461-80.
[3] Kornelyuk AI. Structural and functional investigation of mammalian tyrosyl-tRNA synthetase. Biopolym Cell. 1998; 14(4):349-59.
[4] Korneliuk AI, Kurochkin IV, Matsuka GKh. Tirozil-tRNK-sintetaza iz pecheni byka. Vydelenie i fiziko-khimicheskie svoĭstva. Mol Biol (Mosk). 1988;22(1):176-86.
[5] Gnatenko DV, Korneliuk AI, Kurochkin IV, Ribkinska TA, Matsuka GKh. Vydelenie i kharkteristkia funktsional'no aktivnoĭ proteoliticheski modifitsirovannoĭ formy tirozil-tRNK-sintetazy iz pecheni byka. Ukr Biokhim Zh (1978). 1991;63(4):61-7.
[6] Wakasugi K, Schimmel P. Highly differentiated motifs responsible for two cytokine activities of a split human tRNA synthetase. J Biol Chem. 1999;274(33):23155-9.
[7] Kornelyuk AI, Tas MPR, Dubrovsky AL, Murray CJ. Cytokine activity of the non-catalytic EMAP-2-like domain of mammalian tyrosyl-tRNA synthetasee. Biopolym Cell. 1999; 15(2):168-172.
[8] Guo M, Schimmel P. Essential nontranslational functions of tRNA synthetases. Nat Chem Biol. 2013;9(3):145-53.
[9] Lakowicz JR. Principles of Fluorescence Spectroscopy 3nd ed. Manuscript. Springer New York, NY, 2006; 954 p.
[10] Ladokhin AS. Fluorescence spectroscopy. In Peptide and protein. Chichester, England. John Wiley & Sons Ltd, 2000; 5762-79.
[11] Demchenko AP. Fluorescence and Dynamics in Proteins. In: Eds Lakowicz JR. Topics in Fluorescence Spectroscopy. 2002; 65-111.
[12] Burstein EA, Vedenkina NS, Ivkova MN. Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol. 1973;18(4):263-79.
[13] Chen Y, Barkley MD. Toward understanding tryptophan fluorescence in proteins. Biochemistry. 1998;37(28):9976-82.
[14] Korneliuk AI, Matsuka GKh, Shilin VV. Fluorestsentnyĭ analiz dostupnosti triptofanovykh ostatkov leĭtsil-tRNK-sintetazy v ferment-substratnykh kompleksakh. Biofizika. 1980;25(3):402-4.
[15] Klimenko IV, Guscha TO, Kornelyuk AI. Properties of tryptophan fluorescence of two forms of tyrosyl-tRNA synthetase from bovine liver. Biopolym Cell. 1991;7(6):83-8.
[16] Kornelyuk AI, Klimenko IV, Odynets KA. Conformational change of mammalian tyrosyl-tRNA synthetase induced by tyrosyl adenylate formation. Biochem Mol Biol Int. 1995;35(2):317-22.
[17] Zayets VN, Tsuvarev AYu, Kolomiiets LA, Korneliuk AI. Site-directed mutagenesis of tryptophan residues in the structure of the catalytic module of tyrosyl-tRNA synthetase from Bos taurus. Cytol Genet. 2019; 53(3): 47-57.
[18] Sambrook J, Fritsch T, Manniatis T. Molecular Cloning: A Laboratory Manual. 2th ed. New York: "Cold Spring Harbor Laboratory Press", 1989.
[19] Nishimura A, Morita M, Nishimura Y, Sugino Y. A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucleic Acids Res. 1990;18(20):6169.
[20] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-5.
[21] Kordysh MA, Odynets KA, Kornelyuk AI. Trp144 as a fluorescence probe for investigation of the C-module rapid conformation dynamics in eukaryotic tyrosyle-tRNA synthetase. Biopolym Cell. 2003;19(5):436-9.
[22] Kordysh M, Kornelyuk A. Conformational flexibility of cytokine-like C-module of tyrosyl-tRNA synthetase monitored by Trp144 intrinsic fluorescence. J Fluoresc. 2006;16(5):705-11.
[23] Vivian JT, Callis PR. Mechanisms of tryptophan fluorescence shifts in proteins. Biophys J. 2001;80(5):2093-109.
[24] Royer CA. Probing protein folding and conformational transitions with fluorescence. Chem Rev. 2006;106(5):1769-84.
[25] Engelborghs Y. Correlating protein structure and protein fluorescence. J Fluoresc. 2003; 13: 9-16.
[26] Gallay J, Vincent M, Li de la Sierra IM, Alvarez J, Ubieta R, Madrazo J, Padron G. Protein flexibility and aggregation state of human epidermal growth factor. A time-resolved fluorescence study of the native protein and engineered single-tryptophan mutants. Eur J Biochem. 1993;211(1-2):213-9.
[27] Weitzman C, Consler TG, Kaback HR. Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change. Protein Sci. 1995;4(11):2310-8.
[28] Kozachkov L, Padan E. Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na+/H+ antiporter NhaA. Proc Natl Acad Sci U S A. 2011;108(38):15769-74.
[29] Raghuraman H, Chatterjee S, Das A. Site-Directed Fluorescence Approaches for Dynamic Structural Biology of Membrane Peptides and Proteins. Front Mol Biosci. 2019;6:96.