Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding

Apratim Dhar, Antonios Samiotakis, Simon Ebbinghaus, Lea Nienhaus, Dirar Homouz, Martin Gruebele, Margaret S. Cheung

Research output: Contribution to journalArticle

Abstract

We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function, and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal), and Sph (spherical compact). With an adjustment for viscosity, crowded wild-type PGK and fluorescent PGK are about 15 times or more active in 200 mg/ml Ficoll than in aqueous solution. Our results suggest a previously undescribed solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: Rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates. We also examine T-jump unfolding of PGK as a function of crowding experimentally. We uncover a nonmonotonic folding relaxation time vs. Ficoll concentration. Theory and modeling explain why an optimum concentration exists for fastest folding. Below the optimum, folding slows down because the unfolded state is stabilized relative to the transition state. Above the optimum, folding slows down because of increased viscosity.

Original languageEnglish (US)
Pages (from-to)17586-17591
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number41
DOIs
StatePublished - Oct 12 2010

Keywords

  • Enzymatic activity
  • FRET
  • Folding kinetics
  • Protein conformational changes
  • Thermal denaturation

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding'. Together they form a unique fingerprint.

  • Cite this