New insight in protein-ligand interactions. 2. Stability and properties of two mutant forms of the d-galactose/D-glucose-binding protein from E. coli

Olga V. Stepanenko, Alexander V. Fonin, Olesya V. Stepanenko, Kateryna S. Morozova, Vladislav V. Verkhusha, Irina M. Kuznetsova, Konstantin K. Turoverov, Maria Staiano, Sabato D'Auria

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11 Scopus citations

Abstract

The galactose/glucose-binding protein from E. coli (GGBP) is a 32 kDa protein possessing the typical two-domains structure of the ligand-binding proteins family. GGBP is characterized by low dissociation constant values with respect to glucose binding, displaying an affinity constant for glucose in micromolar range. This feature makes GGBP unsuitable as a sensitive probe for continuous glucose monitoring in blood of diabetic patients. In this work we designed, produced, and characterized two mutant forms of GGBP carrying the following amino acid substitutions in the active center of the protein: W183A or F16A. The two mutant GGBP forms retained a globular structure similar to that of the wild-type GGBP and displayed an affinity for glucose lower than the wild-type GGBP. A deep inspection of the entire set of the obtained results pointed out that the N- and C-terminal domains of GGBP-W183A in the absence of glucose have a stability lower than that of the wild-type protein. In the presence of glucose, the two domains of GGBP-W183A were tightly bound, making the protein structure more stable to the action of denaturing agents. On the contrary, the mutant form GGBP-F16A possesses a very restricted structural stability both in the absence and in the presence of glucose. In this work the role of Phe 16 and W 183 are discussed with regard to the structural and functional features of GGBP. In addition, some general guidelines are reported for the design of a novel glucose biosensor based on the use of GGBP.

Original languageEnglish (US)
Pages (from-to)9022-9032
Number of pages11
JournalJournal of Physical Chemistry B
Volume115
Issue number29
DOIs
Publication statusPublished - Jul 28 2011

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ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

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