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Characterization of Thermotoga maritima maltose binding protein (618.43)
Author(s) -
Masson Laura,
Williams Ashley,
Dattelbaum Jonathan
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.618.43
Subject(s) - thermotoga maritima , maltose binding protein , periplasmic space , thermophile , biochemistry , maltose , circular dichroism , chemistry , dissociation constant , mutant , escherichia coli , fusion protein , enzyme , recombinant dna , gene , receptor
Thermotoga martima is a thermophilic, Gram‐negative bacteria found primarily in hot springs and hydrothermal vents. Its thermophilic characteristics mean that proteins found in this bacterium should be extremely stable, even at very high temperatures, making them ideal for manipulation and biosensor design. The protein of interest for our project is the T. maritima maltose binding protein (TmMBP), which is a member of the periplasmic binding protein superfamily. Using site‐directed mutagenesis of residues in and around the binding pocket, we constructed eight single cysteine mutants of TmMBP. The individual Cys residues we covalently modified with environmentally‐sensitive fluorophores to interrogate maltose binding to the protein. The altered fluorescence characteristics were used to determine a dissociation constant for maltose. We used circular dichroism to evaluate the stability of each mutant. Ultimately, we hope to design a TmMBP biosensor that is stable, can bind molecules other than maltose, and will fluoresce when the target molecule is bound. Grant Funding Source : University of Richmond Arts and Sciences