The use of ion mobility mass spectrometry to assist protein design: a case study on zinc finger fold versus coiled coil interactions

Abstract

The dramatic conformational change in zinc fingers on binding metal ions for DNA recognition makes their structure-function behaviour an attractive target to mimic in de novo designed peptides. Mass spectrometry, with its high throughput and low sample consumption provides insight into how primary amino acid sequence can encode stable tertiary fold. We present here the use of ion mobility mass spectrometry (IM-MS) coupled with molecular dynamics (MD) simulations as a rapid analytical platform to inform de novo design efforts for peptide-metal and peptide-peptide interactions. A dual peptide-based synthetic system, ZiCop based on a zinc finger peptide motif, and a coiled coil partner peptide Pp, have been investigated. Titration mass spectrometry determines the relative binding affinities of different divalent metal ions as Zn(2+) > Co(2+) ≫ Ca(2+). With collision induced dissociation (CID), we probe complex stability, and establish that peptide-metal interactions are stronger and more 'specific' than those of peptide-peptide complexes, and the anticipated hetero-dimeric complex is more stable than the two homo-dimers. Collision cross-sections (CCS) measurements by IM-MS reveal increased stability with respect to unfolding of the metal-bound peptide over its apo-form, and further, larger collision cross sections for the hetero-dimeric forms suggest that dimeric species formed in the absence of metal are coiled coil like. MD supports these structural assignments, backed up by data from visible light absorbance measurements.

Ion Mobility Collision Cross Section Compendium

“…The large blue bubble in Figure 7 corresponds to the 8676 peptide cross sections published by Smith and coworkers in 2010 in support of developing theoretical methods for predicting the IM drift time based upon the primary amino acid sequence. 72 While most of the CCS values have been for tryptic peptides, there is recent and significant efforts being made in the quantitative IM analysis of structurally-interesting peptide and protein classes, including helical peptides, 174–176 metalloproteins, 177–180 intrinsically-disordered proteins, 181–184 metamorphic proteins, 185,186 amyloids, 187–194 and membrane-bound proteins and assemblies. 117,195–198 The last three years has seen a balance of cross section reporting across most of the chemical classes, including lipids and carbohydrates.…”

The multiple conformational charge states of zinc(II) coordination by 2His‐2Cys oligopeptide investigated by ion mobility‐mass spectrometry, density functional theory and theoretical collision cross sections

“…[20] This combination of IM-MS and theoretical modeling provides insight into the multiple coordination structures that are possible between the amb 5 peptide and Zn(II). Computational methods combined with IM-MS have been previously used to characterize a variety of peptide structures including the amyloid peptide using replica exchange molecular dynamics, [21] the alkali metal-bound conformational changes of gramicidin A, [22] the metal-binding to zinc-finger motif peptides, [23,24] and the analysis of Gly-Leu-Gly-Gly-Lys peptide dication structures. [25] Experimental section…”

Deciphering metal ion preference and primary coordination sphere robustness of a designed zinc finger with high‐resolution mass spectrometry

“…In this study, we utilized electrospray ionization mass spectrometry (ESI‐MS), since it allows both the metal ion binding and the resulting conformational transitions to be analyzed simultaneously, without the need for specific structural features (e.g., chromophores), or the use of non‐native metals or chemical labeling . Successful application of high‐resolution ESI‐MS to study metal ion and DNA binding as well as folding of several zinc fingers has been nicely exemplified in a few recent studies …”

“…18 Successful application of high-resolution ESI-MS to study metal ion and DNA binding as well as folding of several zinc fingers has been nicely exemplified in a few recent studies. [19][20][21]…”