Observation of Solvent Penetration during Cold Denaturation of E. coli Phosphofructokinase-2

Ramirez-Sarmiento, CA; Baez, M; Wilson, CAM; Babul, J.; Komives, EA; Guixe, V

Abstract

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N-2 double left right arrow 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 degrees C; unexpectedly, amide exchange increased throughout the protein over time at 3 degrees C. The rate of increase in amide exchange over time at 3 degrees 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.

Más información

Título según WOS: Observation of Solvent Penetration during Cold Denaturation of E. coli Phosphofructokinase-2
Título según SCOPUS: Observation of solvent penetration during cold denaturation of E. coli phosphofructokinase-2
Título de la Revista: BIOPHYSICAL JOURNAL
Volumen: 104
Número: 10
Editorial: Cell Press
Fecha de publicación: 2013
Página de inicio: 2254
Página final: 2263
Idioma: English
URL: http://linkinghub.elsevier.com/retrieve/pii/S0006349513004578
DOI:

10.1016/j.bpj.2013.04.024

Notas: ISI, SCOPUS