Anion-induced folding of rabbit muscle pyruvate kinase: existence of multiple intermediate conformations at low pH.
Structural and functional characteristics of rabbit muscle pyruvate kinase (PK), a tetrameric enzyme having identical subunits, were investigated under neutral as well as acidic conditions by using enzymatic activity measurements and a combination of optical methods, such as circular dichroism, fluorescence, and ANS binding. At low pH and low ionic strength, pyruvate kinase exists in a partially unfolded state (UA state) retaining half of the secondary structure and no tertiary interactions along with a strong binding to the hydrophobic dye, ANS. Addition of anions, like NaCl, KCl, and Na2SO4, to the acid-unfolded state induces refolding, resulting structural propensities similar to that of native tetramer. When anion concentration exceeds a critical limit (0.7 M KCl), a sudden loss of secondary structure and decrease in fluorescence intensity with a redshift in the emission maximum are seen which may be due to the aggregation of the protein, probably due to the intermolecular association. The anion-refolded state is more stable than the UA state, and its stability is nearly equal to that of native protein toward chemical-induced unfolding by Gu-HCl and urea. Moreover, at low concentrations, Gu-HCl behaves like an anion, by inducing refolding of the acid-unfolded state with structural features equivalent to that of native molecule. These observations support a model of protein folding where certain conformations of low free energy prevail and are populated under non-native conditions with different stability.
Edwin
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Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. fedwin@biochem.umass.edu
Jagannadham
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MV
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011770:Pyruvate Kinase / Q000737:chemistry*; D011817:Rabbits; D012997:Solvents; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D012997:Solvents; D014364:Tryptophan; D011770:Pyruvate Kinase; D019791:Guanidine