Fluorescence Resonance Energy Transfer Based Biosensor from Thermophilic Bacterial Periplasmic Binding Protein to Measure Branched-chain and Aromatic Amino Acids
Journal of Advances in Biology & Biotechnology,
Aim: Exploring the ligand binding capacity of leucine-isoleucine-valine binding protein from Thermotoga maritima.
Background: The Fischer ratio, ratio of concentrations between branched-chain and aromatic amino acids, has been considered as an indicator of hepatic disease involving metabolic dysfunctions since its instigation in the 1970's. These amino acids are usually measured by high performance liquid chromatography, gas chromatography and enzymatic spectrophotometry. Considering the future potential of periplasmic binding proteins in clinical diagnosis, leucine-isoleucine-valine binding protein from a thermophilic bacterium Thermotoga maritima was purified and characterized, and evaluated its binding properties with twenty amino acids. The protein's stereo-specificity was also tested due to its importance in astrobiology research. Amino acids and carbohydrates are both known to exist in extraterrestrial environments, and stereo-chemistry is the key aspect for distinguishing between abiotic and biotic origin.
Results: Single amino acid substitutions were generated by overlapping polymerase chain reaction mediated mutagenesis using mutagenic primers to construct two mutants of leucine-isoleucine-valine binding protein (LIVBP). The first mutant of LIVBP in which phenylalanine (F) at 118 position was replaced with cysteine (C) was termed as LIVBP-F118C and the resultant plasmid was named as pKM242. Another mutant of LIVBP in which aspartic acid (D) at 221 position was replaced with cysteine was termed as LIVBP-D221C and the resultant plasmid was named as pKM244. Since the LIVBP-F118C showed higher labeling and fluorescence resonance energy transfer efficiency compared to that of D221C, the LIVBP-F118C was used for further study.
Conclusion: Fluorescence resonance energy transfer technology was applied to measure dissociation constant (Kd) where chimeric isoleucine-valine binding protein (LIVBP) was engineered to conjugate a donor fluorophore at amino-terminal and an acceptor fluorophore at the incorporated cysteine residue. Ligand binding studies revealed that LIVBP from Thermotoga maritima was able to bind with fourteen amino acids out of twenty including branched-chain amino acids (BCAAs) and aromatic amino acids (AAAs) with Kd values ranging from 10-6 to 10-9 M. The Kd values of BCAAs, AAAs, methionine and cysteine were observed at nM level. Highest Kd value was observed for L-leucine (37.2 nM) and L-phenylalanine (44.6 nM). These results indicated that LIVBP from Thermotoga maritima has a broader substrate specificity than previously reported for LIVBP from other organisms, which might be helpful for the development of a miniaturized and noninvasive biosensor to measure BCAAs and AAAs.
- Thermotoga maritima
- fischer ratio
- branched-chain amino acids
- aromatic amino acids
- periplasmic binding proteins
How to Cite
Sun YJ, Rose J, Wang BC, Haiso CD. The structure of glutamine binding protein complexed with glutamine at 1.94A resolution: Comparison with other amino acid binding proteins. J. Mol. Biol. 1998; 278:219-229.
Ames GFL. Bacterial periplasmic transport systems: Structure, mechanism and evolution. Annu. Rev. Biochem. 1986;55: 397-425.
Quiocho FA. Atomic structure of periplasmic binding proteins and the high-affinity active transport systems in bacteria. Phil. Trans. Roy. Soc. B Biol. Sci. 1990; 326:341-351.
de Lorimer RM, Smith JJ, Dwyer MA, Looger LL, Sali KM, Paavola CD, Rizk SS, Sadigov S, Conrad DW, Loew L, Hellinga HW. Construction of a fluorescent biosensor family. Prot. Sci. 2002;11:2655-2675.
Deuschle K, Okumoto S, Fehr M, Looger LL, Kozhukh L, Frommer WB. Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering. Prot. Sci. 2005;14:2304-2314.
Dwyer MA, Hellinga HW. Periplasmic binding proteins: a versatile superfamily for protein engineering. Cyrr. Opin. Struc. Biol. 2004;14:495-504.
Chino S, Sakaguchi A, Yamoto R, Ferri S, Sode K. Branched-chain amino acid biosensing using fluorescent modified engineered leucine/isoleucine/valine binding protein. Int. J. Mol. Sci. 2007; 8:513-525.
Oxender DL, Anderson JJ, Daniels CJ, Landick R, Gunsalus RP, Zurawski G, Selker E, Yanofsky C. Structural and functional analysis of cloned DNA containing genes responsible for branched-chain amino acid transport in Escherichia coli. Proc. Natl. Acad. Sci. USA. 1980;77:1412-1416.
Vorotyntseva TI, Surin AM, Trakhanov SD, Nabiev IR, Antonov VK. Spectral properties of the leucine-isoleucine-valine binding protein and its complexes with substrates. Bioorg. Khim. 1981;7:45-57.
Landick R, Oxender DL. The complete nucleotide sequences of the Escherichia coli LIV-BP and LS-BP genes. Implication for the mechanism of high-affinity branched-chain amino acid transport. J. Biol. Chem. 1985;260:8257-8261.
Koyanagi T, Katayama T, Suzuki H, Kumagai H. Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12. J. Bacteriol. 2004;186:343-350.
Fischer JE, Rosen HM, Ebeid AM, James JH, Keane JM, Soeters PB. The effect of normalization of plasma amino acids on hepatic encephalopathy in man. Surgery. 1976;80:77-91.
Nishitani S, Ijichi C, Takehana K, Fujitani S, Sonaka I. Pharmacological activities of branched-chain amino acids: specificity of tissue and signal transduction. Biochem. Biophys. Res. Commun. 2004;313:387-389.
Nieuwoudt M, Kunnike R, Smuts M, Becker J, Stegmann GF, van der Walt C, Neser J, van der Merwe S. Standardization criteria for an ischemic surgical model of acute hepatic failure in pigs. Biomaterials. 2006;27:3836-3845.
Hughes GJ, Winterhalter KH, Boller W, Wilson KJ. Amino acid analysis using standard high performance liquid chromatography equipment. J. Chromatogr. 1982;235:417-426.
Nanashima A, Yamaguchi H, Shibasaki S, Abo T, Morino S, Yoshinaga M, Sawai T, Tanaka K, Hidaka S, Tsuji T, Nakagoe T, Ayabe H. Changes of branched chain amino acids and tyrosine ratio (BTR) after hepatectomy. Acta. Med. Nagasaki. 2003;48:29-33.
Wu P, Brand L. Resonance energy transfer: Methods and applications. Analytical Biochemistry, 1994;218: 1-13.
Fehr M, Okumoto S, Deuschle K, Lager I, Looger LL, Persson J, Kozhukh L, Lalonde S, Fromer WB. Development and use of fluorescent nanosensors for metabolite imaging in living cells. Biochem. Soc. Trans. 2005;33:287-290.
Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc. Natl. Acad. Sci. USA. 2005;102:8740-8745.
Gu H, Lalonde S, Okumoto S, Looger LL, Scharff-Poulsen AM, Grossman AR, Kossmann J, Jakobsen I, Frommer WB. A novel analytical method for in vivo phosphate tracking. FEBS Lett. 2006;580:5885-5893.
Crochet AP, Kabir MM, Francis MB, Paavola CD. Site-selective dual modification of periplasmic binding proteins for sensing applications. Biosens. Bioelectron. 2010;26:55-61.
Gilmore JM, Scheck RA, Esser-Kahn AP, Joshi NS, Francis MB. N-terminal protein modification through a biomimetic transamination reaction. Angew. Chem. Int. Ed. 2006;45:5307-5311.
Magnusson U, Salopek-Sondi B, Luck LA, Mowbray SL. X-ray structures of the leucine-binding protein illustrate conformational changes and the basis of ligand specificity. J. Biol. Chem. 2004;279:8747-8752.
Hellinga HW, Marvin JS. Protein engineering and the development of generic biosensors. Trends Biotechnol. 1998;16:183-189.
Salins LLE, Ware RA, Ensor CM, Daunert S. A novel reagentless sensing system for measureing glucose based on the glucose/galactose binding protein. Anal. Biochem. 2001;294:19-26.
Karlsson HK, Nilsson PA, Nilsson J, Chibalin AV, Zierath JR, Blomstrand E. Branched-chain amino acids increase p70S6k phosphorylation in human skeletal muscle after resistance exercise. Am. J. Physiol. Endocrinol. Metab. 2004;287:E1-E7.
Blomstrand E, Eliasson J, Karlsson HK, Köhnke R. Branched-chain amino acids activate key enzymes in protein synthesis after physical exercise. J. Nutr. 2006; 136:269S–273S.
Oxender DL, Quay S. Binding proteins and membrane transporter. Annals NY Acad. Sci. 1975; 264:358-372.
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