HOP is a monomer: Investigation of the oligomeric state of the co‐chaperone HOP

• Published in: Protein science 2010-01, Vol.19 (1), p.19-25
• Main Authors: Yi, Fang, Doudevski, Ivo, Regan, Lynne
• Format: Article
• Language: English
• Subjects: analytical ultracentrifugation (AUC); Animals; Area Under Curve; Chromatography, Gel; Drosophila; Drosophila Proteins - chemistry; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Equilibrium; Escherichia coli - genetics; gel filtration chromatography; heat shock organizing protein (HOP); Heat-Shock Proteins - chemistry; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; Histidine - chemistry; Histidine - genetics; Histidine - metabolism; Humans; Molecular weight; Oligopeptides - chemistry; Oligopeptides - genetics; Oligopeptides - metabolism; Protein folding; Protein Multimerization; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; tetratricopeptide repeat (TPR); Ultracentrifugation
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.278

The co‐chaperone Hsp70‐Hsp90 organizing protein (HOP) plays a central role in protein folding in vivo, binding to both Hsp70 and Hsp90 and bringing them together in a functional complex. Reports in the literature concerning the oligomeric state of HOP have been inconsistent—is it a monomer, dimer, or higher order oligomer? Knowing the oligomeric state of HOP is important, because it places limits on the number and types of multiprotein complexes that can form during the folding cycle. Thus, the number of feasible models is simplified. Here, we explicitly investigate the oligomeric state of HOP using three complementary methods: gel filtration chromatography, sedimentation equilibrium analytical ultracentrifugation (AUC), and an in vivo coexpression assay. We find that HOP does not behave like a monomeric globular protein on gel filtration. Rather its behavior is consistent with it being either an elongated monomer or a dimer. We follow‐up on these studies using sedimentation equilibrium AUC, which separates on the basis of molecular weight (MW), independent of shape. Sedimentation equilibrium AUC clearly shows that HOP is a monomer, with no indication of higher MW species. Finally, we use an in vivo coexpression assay that also supports the conclusion that HOP is a monomer.