A series of noncovalent complexes formed between the 86 residue acyl CoA binding protein (ACBP) and a series of acyl CoA derivatives has been studied by electrospray ionization mass spectrometry. Conditions were found under which CoA ligands can be observed in the mass spectrometer bound to ACBP. Despite the very low dissociation constants (10 -7 to 10 -10 M) of the acyl CoA ligand complexes high ratios of ligand-to-protein concentration in the electrospray solution were found to increase the proportion of intact complex observed in the spectrum. Variation in the length of the hydrophobic acyl chain of the ligand (C 16 , C 12 , C 8 , C 0 ) resulted in similar proportions of complex observed in the mass spectrum even though significant variation in solution dissociation constants has been measured. A substantially reduced proportion of complex was, however, found for the mutant proteins, Y28N, Y31N, and Y73F, lacking tyrosine residues involved in critical interactions with the CoA ligand. These results have been interpreted in terms of the different factors stabilizing complexes in the gas phase environment of the mass spectrometer. The complexed species were also investigated by hydrogen-deuterium exchange methods combined with mass spectrometric analysis and the results show that folding of ACBP occurs prior to complex formation in solution. The results also show increased hydrogen exchange protection in the complex when compared with the free protein. Furthermore, even after dissociation of the complex, under these nonequilibrium gas phase exchange conditions, increased protection from hydrogen exchange in the complex is maintained.

The alpha,beta2,mu2,sigma2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu2Thr156-monophosphorylated AP2 mutated in their alphaPtdIns4,5P(2), mu2PtdIns4,5P(2), and mu2Yxxvarphi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxxvarphi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P(2), explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxxvarphi-containing membranes is two step: initial recruitment via the alphaPtdIns4,5P(2) site and then stabilization through the binding of mu2Yxxvarphi and mu2PtdIns4,5P(2) sites to their ligands. The second step is facilitated by a conformational change favored by mu2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxxvarphi binding and is not enhanced by mu2Thr156 phosphorylation.

Recent applications of mass spectrometry (MS) in structural biology have highlighted its ability to define the stoichiometry of numerous protein complexes. [1,2] When combined with tandem MS, this extra dimension has made possible 1) analysis of polydisperse assemblies, [3] 2) characterization of release of proteins from within proteasome and chaperone complexes, [4,5] and 3) identification of proteins released from intact megadalton ribosomes.[6] Tandem MS is effective because macromolecular protein-complex ions decay through a mechanism that involves a dramatic transfer of charge to monomeric subunits prior to their ejection. This asymmetric dissociation acts to distribute the product ion spectrum over a large m/z range, thus enabling separation of ions with overlapping m/z values and identification of heterocomplexes from released subunits.It has been proposed that protein unfolding events are involved in the dissociation mechanism, [7][8][9] although direct evidence pertaining to the structure of the intermediates has not been reported. Ion mobility (IM)-MS, [10,11] a gas-phase technology that separates ions based on their size and shape, has been used to explore gas-phase protein folding, [12][13][14] oligonucleotide structures, [15] and noncovalent complexes. [16][17][18] Herein, we apply IM-MS to examine the activated form of a macromolecular complex. For our studies, we used the 56-kDa complex of tetrameric transthyretin (TTR), primarily because its gas-phase dissociation behavior has been studied extensively.[19] Our experiments were performed on a quadropole-ion mobility-time of flight (Q-IM-ToF) instrument (Synapt, Waters, Milford MA, USA, see the Experimental Section) using an IM separator that employs a series of low-voltage DC waves to push ions through a drift chamber filled with neutral molecules (0.5-1 mBar N 2 ).[20] The speed with which an ion traverses the drift region depends on its collision cross section (CCS); ions with larger CCSs proceed more slowly than ions with smaller ones.These drift times are then calibrated using protein ions of known CCS.[18] Subsequently, molecular modeling is used to generate a range of possible structures, and the CCSs for these models are calculated for comparison with experimental values.Mass spectra for TTR recorded under conditions designed either to maintain or to activate the intact oligomers (80 V or 150 V accelerating voltage, respectively, in the source region of the instrument) are shown (Figure 1 A, C). At 80 V, the minimum accelerating voltage for observation of massresolved protein-complex ions, peaks in the spectrum can be assigned to TTR tetramer and octamer. At 150 V, peaks corresponding to the octamer and tetramer persist; however, under these conditions monomeric subunits are also evident (Figure 1 C). This result indicates that a population of oligomeric TTR ions is undergoing the initial stages of dissociation and is therefore at an ideal stage for analysis of activated states of the complexes.IM data shows that without activation, both tet...

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