new insight in proteinаligand interactions 2 stability and .pdfVIP

ARTICLE pubs.acs.org/JPCB New Insight in ProteinLigand 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,§ and Sabato D’Auria*,§ †Laboratory of Protein structure, stability and folding of proteins, Institute of Cytology RAS, 194064 St. Petersburg, Russia ‡Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, United States §Laboratory for Molecular Sensing, IBP-CNR, 111 80131 Naples, Italy 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-typ