PHOTOINCORPORATION OF 4,4'-BIS(1-ANILINO-8-NAPHTHALENESULFONIC ACID) INTO THE APICAL DOMAIN OF GROEL - SPECIFIC INFORMATION FROM A NONSPECIFIC PROBE

The use of noncovalent hydrophobic probes such as bis-ANS has become i ncreasingly popular in gaining structural information about protein st ructure and conformation. While these probes have provided rich inform ation about protein conformation, specific structural information has been limited. In this report, we extend the usefulness of the probe bi s-ANS by showing that it can be covalently photoincorporated into vari ous proteins. Using the chaperonin GroEL, we have shown that it is pos sible to locate important hydrophobic surfaces through photoincorporat ion and peptide sequencing. It has been proposed that hydrophobic surf aces 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 th e bis-ANS occurs within a 45 residue fragment of the monomer near the middle of the primary sequence. Interestingly, photoincorporation occu rs within this fragment in both tetradecamers and assembly-competent m onomers. From the three-dimensional structure of GroEL, this region ma ps to the apical domain (residues 191-376), which has been implicated in polypeptide binding [Fenton, W. A., Kashi, Y., Furtak, K., and Horw ich, A. L. (1994) Nature 371, 614-619]. In addition, the fluorescent p roperties of the probe are retained including the excitation and emiss ion maxima and the sensitivity to the polarity of its environment. The se results suggest that photoincorporated bis-ANS may be a useful prob e for protein structure and dynamics.