Journal of Protein Chemistry

, Volume 14, Issue 5, pp 299–308

Fluorescence study ofEscherichia coli cyclic AMP receptor protein

  • Marcin Wasylewski
  • Jedrzej Małecki
  • Zygmunt Wasylewski
Article

DOI: 10.1007/BF01886787

Cite this article as:
Wasylewski, M., Małecki, J. & Wasylewski, Z. J Protein Chem (1995) 14: 299. doi:10.1007/BF01886787

Abstract

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76–83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.

Key words

CRP cyclic AMP receptor protein Trp fluorescence circular dichroism Escherichia coli 

Abbreviations

CRP

cyclic AMP receptor protein

NATA

N-acetyltryptophanamide

FQRS

fluorescence-quenching-resolved spectra

FDCD

fluorescence-detected circular dichroism

EDTA

ethylenediaminetetraacetic acid

SDS

sodium dodecyl sulfate

FPLC

fast protein liquid chromatography

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Marcin Wasylewski
    • 1
  • Jedrzej Małecki
    • 1
  • Zygmunt Wasylewski
    • 1
  1. 1.Department of Physical Biochemistry, Institute of Molecular BiologyJagiellonian UniversityKrakówPoland

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