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Fluorescence Resonance Energy Transfer Based Biosensor from Thermophilic Bacterial Periplasmic Binding Protein to Measure Branched-chain and Aromatic Amino Acids

Md. Mohiuddin Kabir, Md. Rayhan Imam, Chowdhury Faiz Hossain, Gazi Nurun Nahar Sultana

Journal of Advances in Biology & Biotechnology · pp. 24–38 · Published 19 Sep 2022

10.9734/jabb/2022/v25i630287

Abstract

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.

FRET Thermotoga maritima biosensor fischer ratio branched-chain amino acids aromatic amino acids periplasmic binding proteins

References (21)

  1. 1 Resonance Energy Transfer: Methods and Applications [DOI]
  2. 2 The effect of normalization of plasma amino acids on hepatic encephalopathy in man.
  3. 3 Branched-Chain Amino Acids Activate Key Enzymes in Protein Synthesis after Physical Exercise [DOI]
  4. 4 Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors [DOI]
  5. 5 N‐Terminal Protein Modification through a Biomimetic Transamination Reaction [DOI]
  6. 6 Periplasmic binding proteins: a versatile superfamily for protein engineering [DOI]
  7. 7 Construction of a fluorescent biosensor family [DOI]
  8. 8 Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering [DOI]
  9. 9 Protein engineering and the development of generic biosensors [DOI]
  10. 10 A novel analytical method for in vivo phosphate tracking [DOI]
  11. 11 X-ray Structures of the Leucine-binding Protein Illustrate Conformational Changes and the Basis of Ligand Specificity [DOI]
  12. 12 Development and use of fluorescent nanosensors for metabolite imaging in living cells [DOI]
  13. 13 Pharmacological activities of branched-chain amino acids: specificity of tissue and signal transduction [DOI]
  14. 14 Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein [DOI]
  15. 15 Site-selective dual modification of periplasmic binding proteins for sensing applications [DOI]
  16. 16 Structural and functional analysis of cloned DNA containing genes responsible for branched-chain amino acid transport in Escherichia coli. [DOI]
  17. 17 Standardization criteria for an ischemic surgical model of acute hepatic failure in pigs [DOI]
  18. 18 N‐Terminal Protein Modification through a Biomimetic Transamination Reaction [DOI]
  19. 19 Bacterial Periplasmic Transport Systems: Structure, Mechanism, and Evolution [DOI]
  20. 20 Changes of Branched Chain Amino Acids and Tyrosine Ratio (BTR) after Hepatectomy
  21. 21 Amino acid analysis using standard high-performance liquid chromatography equipment [DOI]

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