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Comparative Study
. 2015 Jan 20;87(2):1118-26.
doi: 10.1021/ac5037022. Epub 2014 Dec 30.

Amphipols Outperform Dodecylmaltoside Micelles in Stabilizing Membrane Protein Structure in the Gas Phase

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Free PMC article
Comparative Study

Amphipols Outperform Dodecylmaltoside Micelles in Stabilizing Membrane Protein Structure in the Gas Phase

Antonio N Calabrese et al. Anal Chem. .
Free PMC article

Abstract

Noncovalent mass spectrometry (MS) is emerging as an invaluable technique to probe the structure, interactions, and dynamics of membrane proteins (MPs). However, maintaining native-like MP conformations in the gas phase using detergent solubilized proteins is often challenging and may limit structural analysis. Amphipols, such as the well characterized A8-35, are alternative reagents able to maintain the solubility of MPs in detergent-free solution. In this work, the ability of A8-35 to retain the structural integrity of MPs for interrogation by electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is compared systematically with the commonly used detergent dodecylmaltoside. MPs from the two major structural classes were selected for analysis, including two β-barrel outer MPs, PagP and OmpT (20.2 and 33.5 kDa, respectively), and two α-helical proteins, Mhp1 and GalP (54.6 and 51.7 kDa, respectively). Evaluation of the rotationally averaged collision cross sections of the observed ions revealed that the native structures of detergent solubilized MPs were not always retained in the gas phase, with both collapsed and unfolded species being detected. In contrast, ESI-IMS-MS analysis of the amphipol solubilized MPs studied resulted in charge state distributions consistent with less gas phase induced unfolding, and the presence of lowly charged ions which exhibit collision cross sections comparable with those calculated from high resolution structural data. The data demonstrate that A8-35 can be more effective than dodecylmaltoside at maintaining native MP structure and interactions in the gas phase, permitting noncovalent ESI-IMS-MS analysis of MPs from the two major structural classes, while gas phase dissociation from dodecylmaltoside micelles leads to significant gas phase unfolding, especially for the α-helical MPs studied.

Figures

Figure 1
Figure 1
(a) The chemical structure of A8-35, which has an approximate mass of 4 kDa, where x = 29–34%, y = 25–28%, and z = 39–44%. (b) Crystal structures of PagP (left, PDB file 1THQ) and OmpT (right, PDB file 1I78). (c) Crystal structure of Mhp1 (left, PDB file 2X79), and a homology model of GalP (right) based on the crystal structure of XylE which shares 30% sequence identity with GalP.
Figure 2
Figure 2
ESI-IMS-MS data for (a) DDM (red squares) and (b) A8-35 (green diamonds) solubilized PagP acquired under identical instrument parameters (Trap collision energy 100 V). (c) Experimentally determined CCSs of the observed ions at a collision energy of 80 V, with the expected value (based on calculations from the published crystal structure, PDB file 1THQ) indicated by a dotted line. (d) ATDs of PagP (7+ charge state) in DDM and A8-35 at high (dashed lines) and low (solid lines) collision energies. (e) Collision-induced unfolding plot of PagP (7+ charge state) solubilized with DDM and A8-35.
Figure 3
Figure 3
ESI-IMS-MS data for (a) DDM (red squares) and (b) A8-35 (green diamonds) solubilized OmpT acquired under identical instrument parameters (Trap collision energy 180 V). (c) ATDs for the three lowest observed charge states for DDM (solid red line) and A8-35 (dashed green line) solubilized OmpT. (d) Experimentally determined CCSs of the observed ions, with the value based on calculations from the published crystal structure, PDB file 1I78(54) indicated by a dotted line.
Figure 4
Figure 4
ESI-IMS-MS data for DDM and A8-35 solubilized Mhp1 acquired under identical instrument parameters (Trap collision energy 180 V). (a) ESI-IMS-MS spectrum for DDM solubilized Mhp1 (red squares) and (b) ATDs for the three lowest observed charge states (solid red lines). (c) ESI-IMS-MS spectrum for A8-35 solubilized Mhp1 (green diamonds) and (d) ATDs for the three lowest observed charge states (dashed green lines). Peaks corresponding to PE- and CL-bound protein are colored in blue and green, respectively. (e) Experimentally determined CCSs of the observed ions, with the value based on calculations from the published crystal structure, PDB file 2X79(55) indicated by a dotted line.
Figure 5
Figure 5
ESI-IMS-MS data for DDM and A8-35 solubilized GalP acquired under identical instrument parameters (Trap collision energy 180 V). (a) ESI-IMS-MS spectrum for DDM solubilized GalP (red peaks) and (b) ATDs for the three lowest observed charge states (solid red lines). (c) ESI-IMS-MS spectrum for A8-35 solubilized GalP (green diamonds) and (d) ATDs for the three lowest observed charge states (dashed green lines). Peaks corresponding to 2× PE- and 2× CL-bound protein are colored in blue and green, respectively. (e) Experimentally determined CCSs of the DDM (red squares) and A8-35 (green diamonds) solubilized observed GalP ions, with the value based on a model constructed from the published crystal structure of the homologous E. coli glucose transporter XylE indicated by a dotted line.

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