title: Observation of solvent penetration during cold denaturation of E. coli phosphofructokinase-2.
abstract: Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner.
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: César A
authors.initials: CA
authors.lastname: Ramírez-Sarmiento
authors.forename: Mauricio
authors.initials: M
authors.lastname: Baez
authors.forename: Christian A M
authors.initials: CA
authors.lastname: Wilson
authors.forename: Jorge
authors.initials: J
authors.lastname: Babul
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Komives
authors.forename: Victoria
authors.initials: V
authors.lastname: Guixé
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003080:Cold Temperature; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D025481:Phosphofructokinase-2 / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D012997:Solvents; D025481:Phosphofructokinase-2