All MS and DT IM-MS data were acquired on an in-house modified quadropole time-of-flight mass spectrometer (Waters, Manchester, UK) (McCullough et al., 2008) containing a copper drift cell of length 5.1 cm. Nutlin-3 binding to the N-terminal domain of MDM2 (N-MDM2), N-MDM2 presents as at least two conformational families in the absence of Nutlin-3. Upon Nutlin-3 binding, the protein undergoes a compaction event similar to that exhibited by RITA on Rabbit Polyclonal to 5-HT-6 Np53. This multi-technique approach highlights the inherent disorder in these systems; and in particular exemplifies the power of IM-MS as a technique to study transient interactions between small molecule inhibitors and intrinsically disordered proteins. is the ion charge state; is the elementary charge; is the gas number density; is the reduced mass of the ion-neutral pair; is the Boltzmann constant, and is the gas temperature. Here we employ native mass spectrometry, DT IM-MS, circular dichroism (CD) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to observe the conformations of N-terminal p53 domain (Np53) and the N-terminal domain of MDM2 (N-MDM2) both in the gas phase and in solution. We also probe the binding and conformational changes conferred by small molecule inhibitors; Nutlin-3 for N-MDM2, and S55746 hydrochloride RITA for Np53. Further information about DT IM-MS, CD and HDX-MS methodology can be found in the Supporting Information. Materials and methods Expression and purification of both Np53 (residues 1C100) (Szekely et al., 1993; Bakalkin et al., 1995) and N-MDM2 (residues 1C126) (Worrall et al., 2010) have been previously described. Before the analysis reported here, the protein samples were thawed and dialysed in 50 mM ammonium acetate using Bio-RAD micro bio-spin chromatography columns (Bio-Rad Laboratories, Inc.). Concentrations of purified proteins were measured by the Thermo Scientific NanoDrop Spectrophotometer ND 1000 (Thermo Scientific, USA). Small molecule RITA [2,5-bis(5-hydroxymethyl-2-thienyl) furan, NSC 652287] was reconstituted in 100% IPA and stored at ?20C. Before analysis, RITA was thawed and diluted to 100 M and an IPA concentration of 5% using 50 mM ammonium acetate. Nutlin-3 was reconstituted in 100% DMSO and stored at ?80C. Before analysis, Nutlin-3 was thawed and diluted to 500 M and a DMSO concentration of 1% using 50 mM ammonium acetate. MS and IM-MS experiments were performed on Np53 and N-MDM2 from solutions buffered with ammonium acetate (pH 6.8). Np53 samples were incubated with 5% IPA for 30 min at 37C to account for the solvent present in the RITA sample. N-MDM2 S55746 hydrochloride samples were incubated with 0.5% DMSO for 30 min at room temperature to account for the solvent present in the Nutlin-3 sample. Binding experiments were performed on Np53 with RITA in a 1:2 protein:ligand ratio, samples were S55746 hydrochloride incubated for 30 min at 37C. Binding experiments were performed on N-MDM2 and Nutlin-3 in a 1:10 protein:ligand ratio, samples were incubated for 30 min at room temperature. All MS and DT IM-MS data were acquired on an in-house modified quadropole time-of-flight mass spectrometer (Waters, Manchester, UK) (McCullough et al., 2008) containing a copper drift cell of length 5.1 cm. Ions were produced by positive nano-electrospray ionization (nESI) with a spray voltage of 1 1.3C1.62 kV. Helium was used as the buffer gas, its pressure measured using a baratron (MKS Instruments, UK). Buffer gas temperature and pressure readings (294.31C303.69 K and 3.518C3.898 Torr, respectively) were taken at each drift voltage and used in the analysis of drift time measurements. The drift voltage across the cell was varied by decreasing the cell body potential from 60 to 15 V, with arrival time measurements taken at a minimum of five distinct voltages. Instrument parameters were kept as constant as possible and are as follows: cone voltage: 114C119 V, source temperature: 80C. nESI tips were prepared in-house using a micropipette puller (Fleming/Brown model P-97, Sutter Instruments Co., USA) using 4 1.2 mm thin wall glass capillaries (World Precision Instruments, Inc., USA) and filled with 10C20 L of sample. Data was analyzed using MassLynx v4.1 software.