Michael B. Cammarata

Ultraviolet Photodissociation for Characterization of Whole Proteins on a Chromatographic Time Scale.

Intact protein characterization using mass spectrometry thus far has been achieved at the cost of throughput. Presented here is the application of 193 nm ultraviolet photodissociation (UVPD) for top down identification and characterization of proteins in complex mixtures in an online fashion. Liquid chromatographic separation at the intact protein level coupled with fast UVPD and high-resolution detection resulted in confident identification of 46 unique sequences compared to 44 using HCD from prepared Escherichia coli ribosomes. Importantly, nearly all proteins identified in both the UVPD and optimized HCD analyses demonstrated a substantial increase in confidence in identification (as defined by an average decrease in E value of ∼40 orders of magnitude) due to the higher number of matched fragment ions. Also shown is the potential for high-throughput characterization of intact proteins via liquid chromatography (LC)–UVPD-MS of molecular weight-based fractions of a Saccharomyces cerevisiae lysate. In total, protein products from 215 genes were identified and found in 292 distinct proteoforms, 168 of which contained some type of post-translational modification.

Structural Characterization of Dihydrofolate Reductase Complexes by Top-Down Ultraviolet Photodissociation Mass Spectrometry

The stepwise reduction of dihydrofolate to tetrahydrofolate entails significant conformational changes of dihydrofolate reductase (DHFR). Binary and ternary complexes of DHFR containing cofactor NADPH, inhibitor methotrexate (MTX), or both NADPH and MTX were characterized by 193 nm ultraviolet photodissociation (UVPD) mass spectrometry. UVPD yielded over 80% sequence coverage of DHFR and resulted in production of fragment ions that revealed the interactions between DHFR and each ligand. UVPD of the binary DHFR·NADPH and DHFR·MTX complexes led to an unprecedented number of fragment ions containing either an N- or C-terminal protein fragment still bound to the ligand via retention of noncovalent interactions. In addition, holo-fragments retaining both ligands were observed upon UVPD of the ternary DHFR·NADPH·MTX complex. The combination of extensive holo and apo fragment ions allowed the locations of the NADPH and MTX ligands to be mapped, with NADPH associated with the adenosine binding domain of DHFR and MTX interacting with the loop domain. These findings are consistent with previous crystallographic evidence. Comparison of the backbone cleavage propensities for apo DHFR and its holo counterparts revealed significant variations in UVPD fragmentation in the regions expected to experience conformational changes upon binding NADPH, MTX, or both ligands. In particular, the subdomain rotation and loop movements, which are believed to occur upon formation of the transition state of the ternary complex, are reflected in the UVPD mass spectra. The UVPD spectra indicate enhanced backbone cleavages in regions that become more flexible or show suppressed backbone cleavages for those regions either shielded by the ligand or involved in new intramolecular interactions. This study corroborates the versatility of 193 nm UVPD mass spectrometry as a sensitive technique to track enzymatic cycles that involve conformational rearrangements.

Probing the unfolding of myoglobin and domain C of PARP-1 with covalent labeling and top-down ultraviolet photodissociation mass spectrometry.

Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins.

ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair

Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.

Covalent inhibition of new delhi metallo-β-lactamase-1 (NDM-1) by cefaclor

Covalent irreversible inhibitors can successfully treat antibiotic‐resistant infections by targeting serine β‐lactamases. However, this strategy is useless for New Delhi metallo‐β‐lactamase (NDM), which uses a non‐covalent catalytic mechanism and lacks an active‐site serine. Here, NDM‐1 was irreversibly inactivated by three β‐lactam substrates: cephalothin, moxalactam, and cefaclor, albeit at supratherapeutic doses (e.g., cefaclor KI=2.3±0.1 mM; kinact=0.024±0.001 min−1). Inactivation by cephalothin and moxalactam was mediated through Cys208. Inactivation by cefaclor proceeded through multiple pathways, in part mediated by Lys211. Use of a cefaclor metabolite enabled mass spectrometric identification of a +346.0735 Da covalent adduct on Lys211, and an inactivation mechanism is proposed. Lys211 was identified as a promising “handhold” for developing covalent NDM‐1 inhibitors and serves as a conceptual example for creating covalent inhibitors for enzymes with non‐covalent mechanisms.

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates

Pathogenic Escherichia coli, one of the primary causes of urinary tract infections, has shown significant resistance to the most popular antibiotic, trimethoprim (TMP), which inhibits dihydrofolate reductase (DHFR). The resistance is modulated by single point mutations of DHFR. The impact of two clinically relevant mutations, P21L and W30R, on the activity of DHFR was evaluated via measurement of Michaelis–Menten and inhibitory kinetics, and structural characterization was undertaken by native mass spectrometry with ultraviolet photodissociation (UVPD). Compared to WT-DHFR, both P21L and W30R mutants produced less stable complexes with TMP in the presence of co-factor NADPH as evidenced by the relative abundances of complexes observed in ESI mass spectra. Moreover, based on variations in the fragmentation patterns obtained by UVPD mass spectrometry of binary and ternary DHFR complexes, notable structural changes were localized to the substrate binding pocket for W30R and to the M20 loop region as well as the C-terminal portion containing the essential G–H functional loop for the P21L mutant. The results suggest that the mutations confer resistance through distinctive mechanisms. A novel propargyl-linked antifolate compound 1038 was shown to be a reasonably effective inhibitor of the P21L mutant.

A New Covalent Inhibitor of Class C β-Lactamases Reveals Extended Active Site Specificity

O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C β-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C β-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that β-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent β-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor β-lactamases.

Expanding the Scope of Cross-Link Identifications by Incorporating Collisional Activated Dissociation and Ultraviolet Photodissociation Methods

With the advent of new cross-linking chemistries, analytical technologies, and search algorithms, cross-linking has become an increasingly popular strategy for evaluating tertiary and quaternary structures of proteins. Collisional activated dissociation remains the primary MS/MS method for identifications of peptide cross-links in high throughput workflows. Ultraviolet photodissociation (UVPD) at 193 nm has emerged as an alternative ion activation method well-suited for characterization of peptides and has been found in some cases to identify different peptides or provide distinctive sequence information than obtained by collisional activation methods. Complementary high energy collision dissociation (HCD) and UVPD were used in the present study to characterize protein cross-linking for bovine serum albumin, hemoglobin, and E. coli ribosome. Cross-links identified by HCD and UVPD using bis(sulfosuccinimidyl)suberate (BS3), a homobifunctional amine-to-amine cross-linker, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), a heterofunctional amine-to-carboxylic acid cross-linker, were evaluated in the present study. While more unique BS3 cross-links were identified by HCD, UVPD, and HCD provided a complementary panel of DMTMM cross-links which extended the degree of structural insight obtained for the proteins.

Tracking the Catalytic Cycle of Adenylate Kinase by Ultraviolet Photodissociation Mass Spectrometry

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 Mg2+ 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.