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Engineering the maltose binding protein for reagentless fluorescence sensing

G Gilardi1, L Q Zhou, L Hibbert

  • 1Biochemistry Department, Imperial College of Science, Technology and Medicine, London, U.K.

Analytical Chemistry
|November 1, 1994
PubMed
Summary
This summary is machine-generated.

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Researchers engineered a maltose binding protein (MBP) mutant for improved fluorescence sensing. This modification enhances fluorescence upon maltose binding, enabling sensitive detection of this sugar.

Area of Science:

  • Biochemistry and Molecular Biology
  • Protein Engineering
  • Biophysical Chemistry

Background:

  • Maltose binding protein (MBP) is crucial for maltose transport and sensing.
  • Site-directed mutagenesis allows targeted protein modifications.
  • Fluorescent labeling enables real-time monitoring of protein conformational changes.

Purpose of the Study:

  • To create a mutant maltose binding protein (MBP) with altered ligand-binding properties.
  • To develop a novel fluorescence-based sensing strategy for maltose.
  • To investigate protein conformational changes upon ligand binding using fluorescence.

Main Methods:

  • Site-directed mutagenesis to replace serine-337 with cysteine in MBP.
  • Chemical modification of the introduced cysteine residue with fluorophores (IANBD and acrylodan).

Related Experiment Videos

  • Spectroscopic analysis of fluorescence intensity changes upon maltose binding.

    • Main Results:

    • The S337C mutant MBP exhibited a 2-fold reduced affinity for maltose.
    • Fluorescence intensity of IANBD- and acrylodan-labeled MBP increased by 60-180% upon maltose addition.
    • Dissociation constants for maltose were determined to be 62 ± 0.2 µM (IANBD) and 0.8 ± 0.01 µM (acrylodan).

    Conclusions:

    • Combined genetic and chemical modification successfully created a sensitive fluorescent sensor for maltose.
    • The observed fluorescence changes indicate fluorophore burial upon maltose-induced conformational shifts.
    • This strategy holds promise for developing reagentless fluorescence sensing applications.