Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Oct 6:6:8551.
doi: 10.1038/ncomms9551.

Quantifying the stabilizing effects of protein-ligand interactions in the gas phase

Timothy M Allison  1 , Eamonn Reading  1 , Idlir Liko  1 , Andrew J Baldwin  1 , Arthur Laganowsky  1 , Carol V Robinson  1
Affiliations

Quantifying the stabilizing effects of protein-ligand interactions in the gas phase

Timothy M Allison et al. Nat Commun. .

Abstract

The effects of protein-ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. Few translate readily to membrane proteins. We have developed an ion-mobility mass spectrometry approach, which discerns ligand binding to both soluble and membrane proteins directly via both changes in mass and ion mobility, and assesses the effects of these interactions on protein stability through measuring resistance to unfolding. Protein unfolding is induced through collisional activation, which causes changes in protein structure and consequently gas-phase mobility. This enables detailed characterization of the ligand-binding effects on the protein with unprecedented sensitivity. Here we describe the method and software required to extract from ion mobility data the parameters that enable a quantitative analysis of individual binding events. This methodology holds great promise for investigating biologically significant interactions between membrane proteins and both drugs and lipids that are recalcitrant to characterization by other means.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Schematic of the gas-phase protein unfolding experiment and modelling process.
Data shown are for human transthyretin with up to two molecules of L-thyroxine bound, with the unfolding plot generated and modelled for the unbound 15+ ion.
Figure 2
Figure 2. Gas-phase stabilization of soluble and membrane proteins by ligand molecules.
For each system, a typical mass spectrum is shown, with the derived stabilization of the protein by the ligand(s) and a representative crystal structure of the protein. (a) Streptavidin with B4F (stabilization for 16+ charge state, PDB entry 1STP with bound biotin shown as spheres); (b) transthyretin with L-thyroxine (T4) (stabilization for 15+ charge state, PDB entry 2ROX with bound T4 shown as spheres); (c) S. aureus MscL with the lipids L-α-PG, CDL and lysyl-PG (stabilization for 12+ charge state) (structure shown of M. tuberculosis MscL, PDB entry 2OAR). (d) The multidrug and toxic compound extrusion protein from P. furiosus with CDL and PG (stabilization for 11+ charge state, PDB entry 3VVN). Reported are average and s.e.m. from repeated measurements (n=3).
Figure 3
Figure 3. Mass spectrum of the outer membrane protein OmpF from E. coli and gas-phase stabilization by rough lipopolysaccharide (LPS; for 16+ charge state).
The protein structure is of E. coli OmpF (PDB entry 2ZFG). Reported are average and s.e.m. from repeated measurements (n=3).
Figure 4
Figure 4. Comparison of solution- and gas-phase unfolding experiments with two membrane proteins, ammonia channel (AmtB) and aquaporin Z (AqpZ), in the presence of different lipids.
(a) Thermal denaturation (Tm) measurements obtained by DSF of AmtB in the presence of PG, PS and PE at different lipid:protein ratios. Typical raw data are shown for the 10:1 lipid:protein ratio. The colour key applies to the whole figure. (b) Stabilization of AmtB by various lipids calculated using collision-induced unfolding (CIU). (c) Thermal denaturation of AmtB performed by CD in the presence of PG and PE, and (d) of AqpZ in the presence of PE and CDL. Typical raw data showing change in ellipticity at 220 nm are shown for both proteins with the different lipids. (e) Lipid stabilization of AqpZ measured using CIU. Reported are average and s.e.m. from repeated measurements (n=3).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Marcoux J. & Robinson Carol V. Twenty years of gas phase structural biology. Structure 21, 1541–1550 (2013) . - PubMed
    1. Lanucara F., Holman S. W., Gray C. J. & Eyers C. E. The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics. Nat. Chem. 6, 281–294 (2014) . - PubMed
    1. Niu S., Rabuck J. N. & Ruotolo B. T. Ion mobility-mass spectrometry of intact protein–ligand complexes for pharmaceutical drug discovery and development. Curr. Opin. Chem. Biol. 17, 809–817 (2013) . - PubMed
    1. Benesch J. L. P. Collisional activation of protein complexes: picking up the pieces. J. Am. Soc. Mass. Spectrom. 20, 341–348 (2009) . - PubMed
    1. Ruotolo B. T. et al.. Ion mobility-mass spectrometry reveals long-lived, unfolded intermediates in the dissociation of protein complexes. Angew. Chem. Int. Ed. Engl. 46, 8001–8004 (2007) . - PubMed

Publication types

LinkOut - more resources