Temperature modulates binding specificity and affinity of the d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis.

We investigated the effect of temperature on the binding specificity of the recombinant d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). Importantly, we found that TMBP can bind d-glucose (Glc). The Glc binding was characterized by means of fluorescence spectroscopy in the temperature range of 25 degrees C-85 degrees C. Our results show that at 25 degrees C the binding of Glc to TMBP is well represented by a bimodal model with apparent K(d) of 20 muM and approximately 3-8 mM for the first and the second binding step, respectively. At 60 degrees C the binding of Glc to TMBP is represented by a simple hyperbolic model with an apparent K(d) value of about 40 muM. Finally, at 85 degrees C Glc did not bind to TMBP. Molecular dynamics (MD) simulations were used to shed light on the molecular mechanism of the Glc binding. Our results suggest that after proper fluorescent labeling TMBP can be used as a highly thermostable and non-consuming analyte biosensor for monitoring the level of glucose in fluids (e.g. human blood) where other sugars are not present.

[1]  J. Soppa,et al.  Genetic identification of three ABC transporters as essential elements for nitrate respiration in Haloferax volcanii. , 1999, Genetics.

[2]  P. Kollman,et al.  A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules , 1995 .

[3]  C. Sensen,et al.  Glucose Transport in the Extremely Thermoacidophilic Sulfolobus solfataricus Involves a High-Affinity Membrane-Integrated Binding Protein , 1999, Journal of bacteriology.

[4]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[5]  F A Quiocho,et al.  Atomic interactions in protein-carbohydrate complexes. Tryptophan residues in the periplasmic maltodextrin receptor for active transport and chemotaxis. , 1992, Journal of molecular biology.

[6]  E. Schneider,et al.  Biochemical identification of a lipoprotein with maltose-binding activity in the thermoacidophilic Gram-positive bacterium Alicyclobacillus acidocaldarius. , 1996, Research in microbiology.

[7]  Y. J. Sun,et al.  The structure of glutamine-binding protein complexed with glutamine at 1.94 A resolution: comparisons with other amino acid binding proteins. , 1998, Journal of molecular biology.

[8]  J. Lakowicz,et al.  Enzyme fluorescence as a sensing tool: new perspectives in biotechnology. , 2001, Current opinion in biotechnology.

[9]  H. Santos,et al.  The High-Affinity Maltose/Trehalose ABC Transporter in the Extremely Thermophilic Bacterium Thermus thermophilus HB27 Also Recognizes Sucrose and Palatinose , 2005, Journal of bacteriology.

[10]  H. Bahl,et al.  Molecular analysis of the amy gene locus of Thermoanaerobacterium thermosulfurigenes EM1 encoding starch-degrading enzymes and a binding protein-dependent maltose transport system , 1996, Journal of bacteriology.

[11]  K. Diederichs,et al.  The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A. , 2001, Journal of molecular biology.

[12]  H. Santos,et al.  High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis , 1996, Journal of bacteriology.

[13]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[14]  J. DiRuggiero,et al.  Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis , 1998, Journal of bacteriology.

[15]  S. d'Auria,et al.  D‐Trehalose/D‐maltose‐binding protein from the hyperthermophilic archaeon Thermococcus litoralis: The binding of trehalose and maltose results in different protein conformational states , 2006, Proteins.

[16]  Antonio Varriale,et al.  The role of calcium in the conformational dynamics and thermal stability of the D‐galactose/D‐glucose‐binding protein from Escherichia coli , 2005, Proteins.

[17]  E. Gilson,et al.  Evidence for high affinity binding‐protein dependent transport systems in gram‐positive bacteria and in Mycoplasma. , 1988, The EMBO journal.

[18]  Ivo Rendina,et al.  Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis for sugars monitoring , 2006, Extremophiles.