Investigation of GTP-dependent dimerization of G12X K-Ras variants using ultraviolet photodissociation mass spectrometry

Abstract

Variations in backbone cleavage efficiencies during UVPD-MS of G12X variants of K-Ras are used to relate mutation identity to structural changes that impact downstream signaling with Raf.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections

“…Furthermore, full-length farnesylated and methylated KRas (KRas-FMe) does not form dimers by itself on supported membranes in vitro 5 . Paradoxically, indication that Ras functions through dimerization appeared in the early days of Ras research 6 and Ras dimers have been detected at low levels in supported membranes [7][8][9] , in nanodiscs 10 , in cells 11 , in solution by NMR 12 and mass spectrometry 13 . Importantly, dimerization has been shown to be essential for signaling through the Ras/Raf/MEK/ERK pathway in cells and in mice 14 .…”

Elusive structural changes of New Delhi metallo-β-lactamase revealed by ultraviolet photodissociation mass spectrometry

“…Owing to the high energy deposited during UV photoactivation, backbone bonds are cleaved preferentially over the disruption of non-covalent interactions, resulting in production of holo (containing Zn) fragment ions as well as apo (without Zn) fragment ions that elucidate binding sites. 30,[34][35][36]38 For uninhibited NDM-1, holo fragment ions can contain up to two zinc(II) ions, whereas the ebselen-modied protein only has one remaining Zn(II) that can be retained in fragment ions (in addition to the covalently-bound ebselen moiety). Zn-bound holo ions resulting from UVPD were mapped along the protein sequence and upon the crystal structure for uninhibited NDM-1 (Fig.…”

“…† Regions of the protein in which noncovalent interactions or exibility are impacted by single point mutations can be elucidated by examining the enhancement or suppression of UVPD backbone cleavages. [35][36][37] For each NDM variant, the backbone cleavage efficiency upon UVPD was determined and is represented graphically in Fig. S20 † as a function of each residue of the protein.…”

“…31 Additionally, UVPD affords unsurpassed sequence coverage 29,30 and retention of labile covalent moieties 32 with little dependence on precursor charge state. 33 This native UVPD-MS approach has previously been applied for detecting conformational changes induced by ligand binding to dihydrofolate reductase (DHFR), 34 sequence variants of rat sarcoma GTPase K-Ras 35,36 and DHFR, 37 and structural re-organization of the phosphotransferase enzyme adenylate kinase during its catalytic cycle. 38 Here, we use native UVPD-MS to track changes in fragmentation patterns as a means to infer changes in the active site loop conformation, zinc binding, and conformations of surrounding residues in NDM-1 upon binding to three different small molecule inhibitors known to covalently modify the enzyme.…”

Development of a photoinduced fragmentation ion trap for infrared multiple photon dissociation spectroscopy

“…6,14 There are various ion photodissociation schemes used in mass spectrometers besides IRMPD. Examples are UV-visible photodissociation, [15][16][17] infrared and UV-visible [18][19][20] and infrared double resonance experiments 21,22 and blackbody infrared dissociation. 23,24 The main advantages of IRMPD spectroscopy over methods based on PID in other spectral regions [25][26][27][28][29][30][31] is that the IR spectral signatures allow the discrimination of functional groups, the analysis of conformational isomers and the identification of protonation/ coordination sites, especially when supported by theoretical calculations.…”