Monitoring Protein Folding by Mass Spectrometry

Protein folding is key to the structure and function of the protein. The three-dimensional structure dictates its rate of activity and specificity. Ligand binding can change the conformational state of the protein, and changes in the amino acid sequence of the protein can change both the structure and the activity, which may contribute to disease states.

X-ray crystallography and NMR have been two widely used methods for determining three-dimensional structures; however, they have limitations. They require a large amount of purified protein, which is not always possible, and the interpretation of data can be very complicated.¹

Instead, an approach to identify structural information was developed using hydrogen-deuterium exchange and mass spectrometry (HDX).¹ HDX is a powerful tool and is applicable to all proteins because all proteins contain hydrophobic regions with amide bonds.^1,2 The method involves exchanging the exchangeable hydrogens on the protein with deuterium. This involves a mass shift at every point where the exchange has taken place, which can then be used to determine the rate of exchange over time.² Exposing the protein to solvent and causing unfolding allows for the calculation of folding energies. The main difficulty is controlling the back exchange of HDX.¹

In the Gross lab at Washington University in St. Louis, a group of researchers has been working on using HDX for Apolipoprotein E isoforms and their oligomerization.³ Apolipoprotein E (Apo E) is a 34 kDa protein involved in lipid transport from the plasma and CNS and is involved in cholesterol and triglyceride metabolism. There are three major isoforms — ApoE2, ApoE3, and ApoE4 — that are differentiated by single amino-acid changes. Those single amino acids cause significant changes in the biochemical function and properties, as ApoE4 is a risk factor for Alzheimer’s and cardiovascular disease while ApoE2 has a protective role.³ ApoE naturally forms dimers and tetramers depending on the concentration. Unfortunately, like most lipid-bound proteins, data for NMR, X-ray crystallography, and other forms of analysis is complicated.³

The single amino-acid changes in the isoforms of ApoE make it difficult to identify the apolipoprotein being studied, so the group decided to use a Thermo LTQ XL Orbitrap (Thermo Scientific) with ETD for their studies to get the most accurate data possible to differentiate the three isoforms. The idea is to infer the stability of the oligomers, hydrogen bonds, and the solvent accessibility, as well as identifying the spatial resolution by using a digest to resolve the three isoforms. The isoforms themselves prefer to be oligomers, so the rates of exchange would elucidate the regions that are associated with oligomers or are very hydrophobic. The ApoE mixture was solubilized and allowed to exchange with deuterium oxide over a set period of time, at which point the reaction was quenched with acid to maintain the exchanged protons. The proteins were injected into an online pepsin column for digestion and eluted onto a C18 trap to a C18 column through to the mass spectrometer. ETD was used for fragmentation of the peptides for identification of the exchanged protons, as CID scrambles the location of the HDX. Regions that showed slow exchanges were the result of the oligomerization of the ApoE. In particular, slow exchanges were noted for the C-terminal region, which was considered to be significant for oligomerization. As well as identifying and confirming the regions for oligomerization, data were obtained on the differences between the three isoforms showing changes in the C-terminal binding regions, which may explain the differences in the roles of the three isoforms of ApoE. ApoE4, in particular, showed large changes in the rate of exchange, which may be important because the protein is a risk factor for Alzheimer’s and cardiovascular diseases.

The use of HDX to monitor protein folding and unfolding allows vital information to be learned about the three-dimensional protein environment and its interaction with oligomers, small molecules, and the environment, allowing further structure elucidation to be determined on very difficult proteins.

References

1. Liyanage, R., et al. (2012) ‘Theory of the protein equilibrium snapshot by H/D exchange electrospray ionizaton mass spectrometry (PEPS-HDX-ESI-MS) method used to obtain protein folding energies/rates and selected supporting experimental evidence’, International Journal of Mass Spectrometry, 330-332, (pp. 63–70)

2. Burns, K.M., et al. (2013) ‘Platform dependencies in bottom-up hydrogen/deuterium exchange mass spectrometry’, Molecular and Cellular Proteomics, 12 (2), (pp. 539–548)

3. Huang, R.Y.-C., et al. (2012) ‘Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein E oligomerization’, Biochemistry, 50 (43), (pp. 9273–9282)