Detection of an unfolding intermediate in alpha-urease with enhanced affinity for ANSA.
Protein aggregation is believed to be due to conformers that expose hydrophobic clusters that promote protein association. Such conformers can be detected using a fluorescent probe like 8-anilino 1-naphthalenesulfonic acid (ANSA). Here we show that urease exposed to 1.0 M guanidine-hydrogen chloride has a higher affinity for ANSA that native or denatured urease. The binding occurs over a narrow range of denaturant concentration, well below the concentration required to induce denaturation. The impact of ANSA on urease aggregation was further studied by fluorescence, light-scattering, and activity measurements. We found that ANSA modifies urease aggregates and can provide partial protection against inactivation arising from thermally induced aggregation. It seems that the well-known susceptibility of urease to aggregation is due to an intermediate that can be populated in the absence of denaturation. Such a rationale would explain why folding stability of urease is a poor indicator of long-term stabilization by various media.
Peters
C D
CD
Department of Chemistry, University of Prince Edward Island, Charlottetown, Canada.
Walsh
A G
AG
Beauregard
M
M
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D003124:Colorimetry; D005456:Fluorescent Dyes / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014510:Urease / Q000737:chemistry* / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014510:Urease