A partially folded intermediate conformation is induced in pectate lyase C by the addition of 8-anilino-1-naphthalenesulfonate (ANS).
Addition of 8-anilino-1-naphthalenesulfonate (ANS) to acid-denatured pectate lyase C (pelC) leads to a large increase in the fluorescence quantum yield near 480 nm. The conventional interpretation of such an observation is that the ANS is binding to a partially folded intermediate such as a molten globule. Far-ultraviolet circular dichroism demonstrates that the enhanced fluorescence results from the induction of a partially folded protein species that adopts a large fraction of native-like secondary structure on binding ANS. Thus, ANS does not act as a probe to detect a partially folded species, but induces such a species. Near-ultraviolet circular dichroism suggests that ANS is bound to the protein in a specific conformation. The mechanism of ANS binding and structure induction was probed. The interaction of acid-unfolded pelC with several ANS analogs was investigated. The results strongly indicate that the combined effects of hydrophobic and electrostatic interactions account for the relatively high binding affinity of ANS for acid-unfolded pelC. These results demonstrate the need for caution in interpreting enhancement of ANS fluorescence as evidence for the presence of molten globule or other partially folded protein intermediates.
Kamen
D E
DE
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, USA.
Woody
R W
RW
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011133:Polysaccharide-Lyases / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D007527:Isoenzymes; D010940:Plant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D011133:Polysaccharide-Lyases; C023911:pectate lyase