Geldanamycin Alters Abundance and Composition of Low Molecular Weight Proteins and Multiprotein Complexes as Observed by Size Exclusion Column Chromatography

Abstract

Size exclusion chromatography (SEC) is a commonly used and minimally disruptive liquid chromatography‐based technique that separates unbound cytosolic proteins and multiprotein complexes according to their perceived molecular weights. The purpose of this project was to develop a method using SEC to partially purify and isolate biomolecular complexes, including those containing the essential molecular chaperone protein hsp90, in the presence and absence of the hsp90 inhibitor geldanamycin. Using an automated SEC program and S‐200 column, unbound proteins and protein complexes of differing molecular weights were isolated from reticulocyte lysate incubated in the presence and absence of geldanamycin. In addition to hsp90 eluting in a complex of ~250–300 kDa, consistent with it being part of a multiprotein chaperone machinery, other predominant protein elution peaks corresponded to ~26 kDa and ~16 kDa. Interestingly, incubating cell lysate with geldanamycin depleted nearly all of the ~26 kDa peak, indicating an unexpected hsp90 effect. Continued examination of eluate fractions from + and − geldanamycin‐incubated lysates has allowed us to further characterize these geldanamycin‐sensitive low molecular weight proteins as we examine the multitude of intracellular hsp90 functional activities.Support or Funding InformationFunding was provided by the Seattle University College of Science & Engineering undergraduate research program.