Photoincorporation of 4,4'-Bis( 1 -anilino-8-naphthalenesulfonic Acid) into the Apical Domain of GroEL: Specific Information from a Nonspecific Probe

Jeffrey W. Seale, Jennifer L. Martinez, Paul M. Horowitz

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

The use of noncovalent hydrophobic probes such as bis-ANS has become increasingly popular in gaining structural information about protein structure and conformation. While these probes have provided rich information about protein conformation, specific structural information has been limited. In this report, we extend the usefulness of the probe bis-ANS by showing that it can be covalently photoincorporated into various proteins. Using the chaperonin GroEL, we have shown that it is possible to locate important hydrophobic surfaces through photoincorporation and peptide sequencing. It has been proposed that hydrophobic surfaces on the chaperonin may be responsible for the binding of unfolded polypeptides. We show here that photoincorporation of bis-ANS is able to locate a distinct hydrophobic surface on GroEL. Incorporation of the bis-ANS occurs within a 45 residue fragment of the monomer near the middle of the primary sequence. Interestingly, photoincorporation occurs within this fragment in both tetradecamers and assembly-competent monomers. From the three-dimensional structure of GroEL, this region maps to the apical domain (residues 191-376), which has been implicated in polypeptide binding [Fenton, W. A., Kashi, Y., Furtak, K., & Horwich, A. L. (1994) Nature 371, 614-619], In addition, the fluorescent properties of the probe are retained including the excitation and emission maxima and the sensitivity to the polarity of its environment. These results suggest that photoincorporated bis- ANS may be a useful probe for protein structure and dynamics.

Original languageEnglish (US)
Pages (from-to)7443-7449
Number of pages7
JournalBiochemistry
Volume34
Issue number22
DOIs
StatePublished - Jun 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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