Spectroscopic properties of an engineered maltose binding protein

Protein Eng. 1997 May;10(5):479-86. doi: 10.1093/protein/10.5.479.

Abstract

The maltose binding protein (MBP) has been site specifically labelled with a nitrobenzoxadiazole (NBD) group following mutation of a serine to a cysteine residue at position 337. The resulting protein shows a large ligand (maltose or beta-cyclodextrin) dependent increase in its steady-state fluorescence intensity. Analysis of the static (intensity and anisotropy) and dynamic (lifetime distributions) fluorescence of the NBD label as well as the tryptophan residues in both ligand-bound and ligand-free states of this molecule reveals complex multi-component decays that are interpreted in terms of a ligand-induced solvent shielding mechanism. In the context of the known crystal structures of the various forms of the maltose-binding protein (MBP), ligand-dependent changes in both the fluorescence parameters as well as the circular dichroism spectra of the NBD group are interpreted by a twisted intramolecular charge-transfer (TICT) mechanism, wherein ligand binding locks the NBD group into a conformation that prevents efficient relaxation of the excited state.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 4-Chloro-7-nitrobenzofurazan / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Binding Sites
  • Carrier Proteins / chemistry*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Circular Dichroism
  • Crystallography, X-Ray
  • Genetic Engineering*
  • Indicators and Reagents / metabolism
  • Maltose / metabolism*
  • Maltose-Binding Proteins
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Spectrometry, Fluorescence

Substances

  • Bacterial Proteins
  • Carrier Proteins
  • Indicators and Reagents
  • Maltose-Binding Proteins
  • Membrane Proteins
  • Maltose
  • 4-Chloro-7-nitrobenzofurazan