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 increasingly popular in gaining structural information about protein structure and conformation. While these probes have provided rich information about protein conformation, specific 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.
Seale
J W
JW
Department of Biochemistry, University of Texas Health Sciences Center at San Antonio 78240-7760, USA.
Martinez
J L
JL
Horowitz
P M
PM
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D005456:Fluorescent Dyes; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry; D014466:Ultraviolet Rays; D014508:Urea
D000817:Anilino Naphthalenesulfonates; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D010446:Peptide Fragments; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014508:Urea; D013884:Thiosulfate Sulfurtransferase