Structural Studies of a Stapled Peptide with Native Ion Mobility-Mass Spectrometry and Transition Metal Ion Förster Resonance Energy Transfer in the Gas Phase

Abstract

Native mass spectrometry has emerged as an important tool for gas-phase structural biology. However, the conformations that a biomolecular ion adopts in the gas phase can differ from those found in solution. Herein, we report a synergistic, native ion mobility-mass spectrometry (IM-MS) and transition metal ion Forster resonance energy transfer (tmFRET)based approach to probe the gas-phase ion structures of a nonstapled peptide (nsp; Ac-CAARAAHAAAHARARA-NH 2 ) and a stapled peptide (sp; Ac-CXARAXHAAAHARARA-NH 2 ). The stapled peptide contains a single hydrocarbon chain connecting the peptide backbone in the i and i + 4 positions via a Grubbs ring-closure metathesis. Fluorescence lifetime measurements indicated that the Cu-bound complexes of carboxyrhodamine 6g (crh6g)-labeled stapled peptide (sp-crh6g) had a shorter donor−acceptor distance (r DA ) than the labeled nonstapled peptide (nsp-crh6g). Experimental collision cross-section (CCS) values were then determined by native IM-MS, which could separate the conformations of Cu-bound complexes of nsp-crh6g and sp-crh6g. Finally, the experimental CCS (i.e., shape) and r DA (i.e., distance) values were used as constraints for computational studies, which unambiguously revealed how a staple reduces the elongation of the peptide ions in the gas phase. This study demonstrates the superiority of combining native IM-MS, tmFRET, and computational studies to investigate the structure of biomolecular ions.

The Structure of Cyclic Neuropeptide Somatostatin and Octapeptide Octreotide in the Presence of Copper Ions: Insights from Transition Metal Ion FRET and Native Ion Mobility-Mass Spectrometry

“…Then, FRET efficiencies (E FRET ) and donor−acceptor distances (r DA ), defined as the distance between cR6G and Cu(II) ions, were calculated (Figure 1e) with a Forster distance (R 0 ) in the gas phase equal to 26.0 Å. 25 The results show a smaller calculated r DA value (∼18 Å) and a larger calculated r DA value (∼40 Å) for both F 1 and K 5 . Typically, this finding would suggest the presence of extended and compact conformational populations in linear peptides.…”

“…This suggests that reduction of the donor lifetime in the presence of copper results from FRET and not from other photophysical or structural effects. Then, FRET efficiencies ( E FRET ) and donor–acceptor distances ( r DA ), defined as the distance between cR6G and Cu(II) ions, were calculated (Figure e) with a Förster distance ( R 0 ) in the gas phase equal to 26.0 Å . The results show a smaller calculated r DA value (∼18 Å) and a larger calculated r DA value (∼40 Å) for both F 1 and K 5 .…”

“…17−19 Combining native MS and FRET has emerged as a useful approach to measure intramolecular distances of mass-selected biomolecular ions. 20−23 Furthermore, transition metal ion FRET (tmFRET), a recent expansion of gas-phase FRET, 24,25 utilizes metal ions (e.g., copper, nickel, or cobalt) for fluorescence quenching to probe shorter distances (10−40 Å) compared to traditional FRET (20−100 Å). Thus, the Cu(II) ions bound to SST can function directly as an acceptor and obviates the need for another labeling step, which could lead to an increased structural perturbation of the native SST structure.…”

“…The combination of global shape (IM-MS) and intramolecular distance (tmFRET) constraints facilitates an orthogonal interpretation of gas-phase structures. 25,26 Here, we make use of gas-phase methods to isolate ions of well-defined charge state and constitution to report on (i) the capability of tmFRET to localize Cu(II) ion binding sites in SST and OCT, (ii) structural information on OCT and SST utilizing multiple labeling isomers (tmFRET) and IM-MS, and (iii) whether the structural information obtained can explain some of the biological functions of SST and OCT when Cu(II) ions are bound.…”

CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules

“…So, increasing the mass or volume of fluorescent molecule is often used to amplify the FA signal. In previous reports, the volume and mass are usually enhanced by inorganic nanomaterials. − However, the strong fluorescence resonance energy transfer (FRET) − or electron transfer (ET) − between inorganic materials and fluorescence groups greatly reduces the accuracy of the FA values. Recently, biomacromolecules are found to have excellent FA amplification abilities without a fluorescence quenching effect. − ,− Especially, our group successfully used DNA nanosheets (DNSs) to amplify FA for the detection of biomolecules .…”