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Detection of a protein conformational equilibrium by electrospray ionisation-ion mobility-mass spectrometry.

Ion mobility spectrometry (IMS) is emerging as a promising technique for providing low-resolution protein-structure information, particularly in combination with electrospray ionization (ESI) and mass spectrometry (MS). There is currently much debate on the structure of protein ions in the gas phase, as summarized by Breuker and McLafferty. Whilst it appears probable that some structural collapse occurs within picoseconds of dehydration, the onset of gross structural rearrangement may require tens of milliseconds. This time provides a potential window for the observation of “near-native” structures that may retain some elements of the solution structure, with the ability to provide biologically relevant information. A number of recent applications have used IMS to study the conformation and stoichiometry of proteins and their complexes. Structural changes to amyloid and prion proteins, as well as steps in amyloid fibril assembly, have been detected by using this approach, and the calcium-dependent conformational change in calmodulin has been probed by IM-MS, as has the relationship between tertiary structure and chemotactic activity in antibacterial peptides. Insights into the structures of large multiprotein complexes, such as the RNA-binding TRAP protein, GroEL, and the 20S proteasome have also been provided by ion mobility measurements. We postulated that IM-MS may be used to study the dynamic equilibrium between well-characterized conformations of a monomeric multidomain protein. To test this hypothesis, we have examined NADPH-cytochrome P450 reductase (CPR) using ESI-IM-MS. CPR is a 76 kDa membrane bound flavoprotein that catalyses the transfer of electrons from NADPH to a number of oxygenase enzymes. CPR consists of three folded domains, an FADand NADPH-binding domain, an FMN-binding domain, and a linker domain which may serve to orient the other two domains. The FMN-binding domain is connected to the rest of the protein by a 14-residue “hinge”, thus providing the flexibility that is thought to be important for the function of the protein. An N-terminal 57 amino acid peptide is responsible for anchoring CPR to the endoplasmic reticulum membrane; recombinant CPR, which lacks this N-terminal peptide, is both soluble and functional, thus facilitating detailed structure–activity studies. The CPR-mediated electron transfer from NADPH to cytochrome P450 proceeds in a stepwise fashion: NADPH! FAD!FMN!P450. Interflavin electron transfer requires spatial proximity of the two prosthetic groups, and the X-ray crystal structure of CPR (PDB file: 1AMO) confirms this is the case (closest approach of the FAD and FMN methyl groups: 3.85 (C–C)). However, in this compact or “closed” conformation, the FMN cofactor appears to be inaccessible to the large cytochrome P450 molecule, and so the need for domain movement as an essential part of the catalytic cycle has been widely assumed. Recently, NMR spectroscopy, small-angle X-ray scattering (SAXS), and crystallographic evidence for this movement has been obtained, thus suggesting that in solution, CPR exists in an equilibrium between a compact conformation appropriate for interflavin electron transfer and an extended conformation appropriate for electron transfer to P450 (Figure 1). Herein we show that two major conformations of wildtype CPR are present in the gas phase, and that their relative abundance can be influenced by the ionic strength of the solution from which they are electrosprayed, by removal of key intramolecular ionic interactions, and, crucially, by the redox state of the flavin groups. This study demonstrates the ability of ESI-IMS-MS to detect a protein conformational