Denaturation and urea gradient gel electrophoresis of arginine kinase: evidence for a collapsed-state conformation.
The unfolding transition of monomeric arginine kinase from shrimp was examined in a multiparameter equilibrium approach. Parameters investigated included catalytic activity, circular dichroism, intrinsic fluorescence characteristics including acrylamide quenching, and steady-state anisotropy of arginine kinase derivatized at the reactive cysteine with fluorescent dye 5-[[[(iodoacetyl)amine]ethyl]amino]-naphthalene-1-sulfonic acid. The time course of electrophoretic patterns in urea gradient gels was also determined. Midpoints of the transitions varied considerably depending upon the parameter, indicating the presence of populated intermediates. Significant unfolding began after 2 M urea with most secondary and tertiary structure eliminated in 5 M urea. In dilute denaturant, arginine kinase exhibited a small increase in specific activity and physical properties characteristic of a protein with collapsed structure, including an increase in alpha-helical content, a decrease in intrinsic fluorescence (without a shift in the emission maximum), an increase in anisotropy, and a decrease in fractional accessibility by tryptophan to acrylamide quenching. The electrophoretic pattern of arginine kinase in urea gradient gels is consistent with the presence of a compact conformation in dilute denaturant. The results indicate the existence of a contracted overall conformation in dilute urea. The persistence of catalytic activity suggests this structure may be a functional molecular isoform, but the obvious differences in structure between the native state and the conformation of arginine kinase in 0.5 M urea raise the question of whether such isoforms may also be a type of folding intermediate.
France
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RM
Department of Chemistry, University of South Florida, Tampa 33620, USA.
Grossman
S H
SH
D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry* / Q000302:isolation & purification; D002942:Circular Dichroism; D033364:Decapoda / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D011487:Protein Conformation; D014508:Urea
D014508:Urea; D001122:Arginine Kinase