Single-residue mutations at Gly12 (G12X) in the GTP-ase protein K-Ras can lead to activation of different downstream signaling pathways, depending on the identity of the mutation, through a poorly defined mechanism. Herein, native mass spectrometry combined with top-down ultraviolet photodissociation (UVPD) was employed to investigate the structural changes occurring from G12X mutations of K-Ras. Complexes between K-Ras or the G12X mutants and guanosine 5'-diphosphate (GDP) or GDPnP (a stable GTP analogue) were transferred to the gas phase by nano-electrospray ionization and characterized using UVPD. Variations in the efficiencies of backbone cleavages were observed upon substitution of GDPnP for GDP as well as for the G12X mutants relative to wild-type K-Ras. An increase in the fragmentation efficiency in the segment containing the first 50 residues was observed for the K-Ras/GDPnP complexes relative to the K-Ras/GDP complexes, whereas a decrease in fragmentation efficiency occurred in the segment containing the last 100 residues. Within these general regions, the specific residues at which changes in fragmentation efficiency occurred correspond to the phosphate and guanine binding regions, respectively, and are indicative of a change in the binding motif upon replacement of the ligand (GDP versus GDPnP). Notably, unique changes in UVPD were observed for each G12X mutant with the cysteine and serine mutations exhibiting similar UVPD changes whereas the valine mutation was significantly different. These findings suggest a mechanism that links the identity of the G12X substitution to different downstream effects through long-range conformational or dynamic effects as detected by variations in UVPD fragmentation.
RNA polymerase II contains a repetitive, intrinsically disordered, C-terminal domain (CTD) composed of heptads of the consensus sequence YSPTSPS. The CTD is heavily phosphorylated and serves as a scaffold, interacting with factors involved in transcription initiation, elongation and termination, RNA processing and chromatin modification. Despite being a nexus of eukaryotic gene regulation, the structure of the CTD and the structural implications of phosphorylation are poorly understood. Here we present a biophysical and biochemical interrogation of the structure of the full length CTD of Drosophila melanogaster, which we conclude is a compact random coil. Surprisingly, we find that the repetitive CTD is structurally heterogeneous. Phosphorylation causes increases in radius, protein accessibility and stiffness, without disrupting local structural heterogeneity. Additionally, we show the human CTD is also structurally heterogeneous and able to substitute for the D. melanogaster CTD in supporting fly development to adulthood. This finding implicates conserved structural organization, not a precise array of heptad motifs, as important to CTD function.
The complex interplay of dynamic protein plasticity and specific side-chain interactions with substrate molecules that allows enzymes to catalyze reactions has yet to be fully unraveled. Top-down ultraviolet photodissociation (UVPD) mass spectrometry is used to track snapshots of conformational fluctuations in the phosphotransferase adenylate kinase (AK) throughout its active reaction cycle by characterization of complexes containing AK and each of four different adenosine phosphate ligands. Variations in efficiencies of UVPD backbone cleavages were consistently observed for three α-helices and the adenosine binding regions for AK complexes representing different steps of the catalytic cycle, implying that these stretches of the protein sample various structural microstates as the enzyme undergoes global open-to-closed transitions. Focusing on the conformational impact of recruiting or releasing the Mg cofactor highlights two loop regions for which fragmentation increases upon UVPD, signaling an increase in loop flexibility as the metal cation disrupts the loop interactions with the substrate ligands. Additionally, the observation of holo ions and variations in UVPD backbone cleavage efficiency at R138 implicate this conserved active site residue in stabilizing the donor phosphoryl group during catalysis. This study showcases the utility of UVPD-MS to provide insight into conformational fluctuations of single residues for active enzymes.
Unraveling disease mechanisms requires a comprehensive understanding of how the interplay between higher-order structure and protein-ligand interactions impacts the function of a given protein. Recent advances in native mass spectrometry (MS) involving multimodal or higher-energy activation methods have allowed direct interrogation of intact protein complexes in the gas phase, allowing analysis of both composition and subunit connectivity. We report a multistage approach combining collisional activation and 193 nm ultraviolet photodissociation (UVPD) to characterize single amino acid variants of the human mitochondrial enzyme branched-chain amino acid transferase 2 (BCAT2), a protein implicated in chemotherapeutic resistance in glioblastoma tumors. Native electrospray ionization confirms that both proteins exist as homodimers. Front-end collisional activation disassembles the dimers into monomeric subunits that are further interrogated using UVPD to yield high sequence coverage of the mutated region. Additionally, holo (ligand-bound) fragment ions resulting from photodissociation reveal that the mutation causes destabilization of the interactions with a bound cofactor. This study demonstrates the unique advantages of implementing UVPD in a multistage MS approach for analyzing intact protein assemblies.
With an overarching goal of characterizing the structure of every protein within a cell, identifying its interacting partners, and quantifying the dynamics of the states in which it exists, key developments are still necessary to achieve comprehensive native proteomics by mass spectrometry (MS). In practice, much work remains to optimize reliable online separation methods that are compatible with native MS and improve tandem MS (MS/MS) approaches with respect to when and how energy is deposited into proteins of interest. Herein, we utilize native capillary zone electrophoresis coupled with MS to characterize the proteoforms in the Escherichia coli 70S ribosome. The capabilities of 193 nm ultraviolet photodissociation (UVPD) to yield informative backbone sequence ions are compared to those of higher-energy collisional dissociation (HCD). To further improve sequence coverage values, a multistage MS/MS approach is implemented involving front-end collisional activation to disassemble protein complexes into constituent subunits that are subsequently individually isolated and activated by HCD or UVPD. In total, 48 of the 55 known E. coli ribosomal proteins are identified as 84 unique proteoforms, including 22 protein–metal complexes and 10 protein–protein complexes. Additionally, mapping metal-bound holo fragment ions resulting from UVPD of protein–metal complexes offers insight into the metal-binding sites.
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