Electron capture dissociation and drift tube ion mobility-mass spectrometry coupled with site directed mutations provide insights into the conformational diversity of a metamorphic protein.

Sophie R Harvey,Robert C Tyler,Perdita E Barran,Massimiliano Porrini,Cait E Macphee,Brian F Volkman

doi:10.1039/c4cp05136j

Abstract

Ion mobility mass spectrometry can be combined with data from top-down sequencing to discern adopted conformations of proteins in the absence of solvent. This multi-technique approach has particular applicability for conformationally dynamic systems. Previously, we demonstrated the use of drift tube ion mobility-mass spectrometry (DT IM-MS) and electron capture dissociation (ECD) to study the metamorphic protein lymphotactin (Ltn). Ltn exists in equilibrium between distinct monomeric (Ltn10) and dimeric (Ltn40) folds, both of which can be preserved and probed in the gas-phase. Here, we further test this mass spectrometric framework, by examining two site directed mutants of Ltn, designed to stabilise either distinct fold in solution, in addition to a truncated form consisting of a minimum model of structure for Ltn10. The truncated mutant has similar collision cross sections to the wild type (WT), for low charge states, and is resistant to ECD fragmentation. The monomer mutant (CC3) presents in similar conformational families as observed previously for the WT Ltn monomer. As with the WT, the CC3 mutant is resistant to ECD fragmentation at low charge states. The dimer mutant W55D is found here to exist as both a monomer and dimer. As a monomer W55D exhibits similar behaviour to the WT, but as a dimer presents a much larger charge state and collision cross section range than the WT dimer, suggesting a smaller interaction interface. In addition, ECD on the W55D mutant yields greater fragmentation than for the WT, suggesting a less stable β-sheet core. The results highlight the power of MS to provide insight into dynamic proteins, providing further information on each distinct fold of Ltn. In addition we observe differences in the fold stability following single or double point mutations. This approach, therefore, has potential to be a useful tool to screen for the structural effects of mutagenesis, even when sample is limited.

Highlights

The development of ‘soft’ ionisation techniques such as electrospray (ESI)[1,2] and nano-electrospray ionisation (n-ESI)[3,4] revolutionised biological mass spectrometry (MS)
Initial studies focused on a truncated Ltn mutant (WT 1–72), which contains the structural core of the protein but does not contain the last 21 amino acids which form an intrinsically disordered tail in the wild type (WT) protein
In addition at the [M1–72 + 7H]7+ and [M1–72 + 8H]8+ charge states we observe an increase in the fragmentation in the a-helical region, for both c and z type fragments in addition to zIc fragments, suggesting this region is beginning to unravel from the structural core of the protein, losing any non-covalent stabilising interactions. This observation is consistent with our DT ion mobility-mass spectrometry (IM-MS) observations in which we see a significant increase in collision cross section (CCS) for the [M1–72 + 7H]7+ and [M1–72 + 8H]8+ species

Summary

Introduction

The development of ‘soft’ ionisation techniques such as electrospray (ESI)[1,2] and nano-electrospray ionisation (n-ESI)[3,4] revolutionised biological mass spectrometry (MS) It is accepted as a powerful tool in the structural analysis of proteins and protein complexes that can probe solution-phase topologies and even in vivo active structures.[5,6,7,8,9,10] In the early 1960s the first reports of combining the technique of ion mobility spectroscopy (IMS) to mass spectrometry were published.[11,12,13] This hybrid technique known as ion mobility-mass spectrometry (IM-MS) provides an extra dimension of information over mass spectrometry alone, separating ions based on their mass-tocharge ratio and on their size and shape, known as their rotationally averaged collision cross section (CCS). The results provide detailed insight into the unfolding of each distinct Ltn conformation, in conjunction with studying the effect of specific site mutations on both folds and allowing any subsequent increase or decrease in fold stability to be identified

Results and discussion

Conclusions

Experimental procedures