Thermodynamic Analysis of the Geldanamycin–Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach

Abstract

Abstract. Geldanamycin is a natural product with well-established and potent anticancer activities. Heat shock protein 90 (Hsp90) is the known target of geldanamycin, which directly binds to Hsp90's N-terminal ATP binding domain and inhibits Hsp90's ATPase activity. The affinity of geldanamycin for Hsp90 has been measured in multiple studies. However, there have been large discrepancies between the reported dissociation constants (i.e., K d values), which have ranged from low nanomolar to micromolar. Here the stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to measure the binding affinity of geldanamycin to unpurified Hsp90 in an MCF-7 cell lysate. The K d values determined here were dependent on how long geldanamycin was equilibrated with the lysate prior to SPROX analysis. The K d values determined using equilibration times of 0.5 and 24 h were 1 and 0.03 μM, respectively. These K d values, which are similar to those previously reported in a geldanamycinHsp90 binding study that involved the use of a fluorescently labeled geldanamycin analogue, establish that the slow-tight binding behavior previously observed for the fluorescently labeled geldanamycin analogue is not an artifact of the fluorescent label, but rather an inherent property of the geldanamycin-Hsp90 binding interaction. The slow-tight binding property of this complex may be related to time-dependent conformational changes in Hsp90 and/or to time-dependent chemical changes in geldanamycin, both of which have been previously proposed to explain the slow-tight binding behavior of the geldanamycin-Hsp90 complex.

Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale

“…These two model drugs were chosen for these proof-of-principle studies because the binding interactions with their protein targets have been well-studied by conventional methods and by other energetic-based studies, such as chemical denaturation and protein precipitation (CPP), SPROX and SILAC-PP methodology. 10, 15–19…”

“…6 The proteomic coverages observed in these STEPP-PP experiments were also significantly greater (~40% larger) than the proteomic coverage observed in similar drug-mode-of action study using SPROX to identify protein targets in the yeast proteome. 19…”

Proteome-Wide Structural Biology: An Emerging Field for the Structural Analysis of Proteins on the Proteomic Scale

“…In SPROX and PP this chemical denaturant concentration can be used to calculate thermodynamic parameters such as a protein folding free energy or the dissociation constant of a protein-ligand complex. 47, 48, 63–65…”

Global analysis of methionine oxidation provides a census of folding stabilities for the human proteome

“…Furthermore, the use of chemical denaturants prevents the irreversible aggregation of proteins during the course of denaturation—a phenomenon that typically confounds thermal unfolding experiments. To date, SPROX has been used to analyze the stability of individual intact proteins and to examine proteome-wide alterations in denaturation patterns induced by ligand binding (20, 22, 23). Here, we have coupled SPROX with highly multiplexed tandem mass tagging (TMT) to obtain high-resolution denaturation curves—an approach we have termed high-resolution SPROX (HR-SPROX).…”