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IntroductionType 2 diabetes mellitus (T2D) confers high atherosclerotic cardiovascular disease (ASCVD) risk. The metabolite trimethylamine N-oxide (TMAO) derived via gut flora has been linked to excess ASCVD.Research design and methodsWe analyzed data, biospecimens, and major adverse cardiovascular events (MACEs) from the prospective multicenter randomized Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial to assess its value in 330 high-risk individuals with T2D without evident atherosclerotic disease at enrollment.ResultsIncident cardiovascular events occurred in 165 cases; 165 controls matched by age, sex, and treatment arm experienced no incident events during follow-up. Cases and controls (mean age 64.5 years) had similar mean glycated hemoglobin (HbA1c) (8.2%) and mean 10-year ASCVD risk (23.5%); groups also had similar use of statins and antihypertensive medications at baseline and follow-up. Baseline plasma TMAO levels did not differ between groups after adjusting for ASCVD risk score, HbA1c, and estimated glomerular filtration rate, nor did TMAO distinguish patients suffering incident MACE from those who remained event-free.ConclusionsTMAO’s prognostic value for incident ASCVD events may be blunted when applied to individuals with T2D with poor glycemic control and high baseline ASCVD risk. These results behoove further translational investigations of unique mechanisms of ASCVD risk in T2D.

Biosynthesis of the peptidyl nucleoside antifungal agent blasticidin S in Streptomyces griseochromogenes requires the hydrolytic function of a nucleotide hydrolase, BlsM, to excise the free cytosine from the 5'-monophosphate cytosine nucleotide. In addition to its hydrolytic activity, interestingly, BlsM has also been shown to possess a novel cytidine deaminase activity, converting cytidine, and deoxycytidine to uridine and deoxyuridine. To gain insight into the substrate specificity of BlsM and the mechanism by which it performs these dual function, the solution structure of BlsM was determined by multi-dimensional nuclear magnetic resonance approaches. BlsM displays a nucleoside deoxyribosyltransferase-like dimeric topology, with each monomer consisting of a five-stranded β-sheet that is sandwiched by five α-helixes. Compared with the purine nucleotide hydrolase RCL, each monomer of BlsM has a smaller active site pocket, enclosed by a group of conserved hydrophobic residues from both monomers. The smaller size of active site is consistent with its substrate specificity for a pyrimidine, whereas a much more open active site, as in RCL might be required to accommodate a larger purine ring. In addition, BlsM confers its substrate specificity for a ribosyl-nucleotide through a key residue, Phe19. When mutated to a tyrosine, F19Y reverses its substrate preference. While significantly impaired in its hydrolytic capability, F19Y exhibited a pronounced deaminase activity on CMP, presumably due to an altered substrate orientation as a result of a steric clash between the 2'-hydroxyl of CMP and the ζ-OH group of F19Y. Finally, Glu105 appears to be critical for the dual function of BlsM.

To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).

Salmonellais a foodborne pathogen that causes annually millions of cases of salmonellosis globally, yet Salmonella-specific antibacterials are not available. During inflammation, Salmonella utilizes the Amadori compound fructose-asparagine (F-Asn) as a nutrient through the successive action of three enzymes, including the terminal FraB deglycase. Salmonella mutants lacking FraB are highly attenuated in mouse models of inflammation due to the toxic build-up of the substrate 6-phosphofructose-aspartate (6-P-F-Asp). This toxicity makes Salmonella FraB an appealing drug target, but there is currently little experimental information about its catalytic mechanism. Therefore, we sought to test our postulated mechanism for the FraB-catalyzed deglycation of 6-P-F-Asp (via an enaminol intermediate) to glucose-6-phosphate and aspartate. A FraB homodimer model generated by RosettaCM was used to build substrate-docked structures that, coupled with sequence alignment of FraB homologs, helped map a putative active site. Five candidate active-site residues-including three expected to participate in substrate binding-were mutated individually and characterized. Native mass spectrometry and ion mobility were used to assess collision cross sections and confirm that the quaternary structure of the mutants mirrored the wild type, and that there are two active sites/homodimer. Our biochemical studies revealed that FraB Glu214Ala, Glu214Asp, and His230Ala were inactive in vitro, consistent with deprotonated-Glu214 and protonated-His230 serving as a general base and a general acid, respectively. Glu214Ala or His230Ala introduced into the Salmonella chromosome by CRISPR/Cas9-mediated genome editing abolished growth on F-Asn. Results from our computational and experimental approaches shed light on the catalytic mechanism of Salmonella FraB and of phosphosugar deglycases in general.

SUMMARY The Ebola virus matrix protein VP40 forms distinct structures linked to distinct functions in the virus life cycle. Dimeric VP40 is a structural protein associated with virus assembly, while octameric, ring-shaped VP40 is associated with transcriptional control. In this study, we show that suitable nucleic acid is sufficient to trigger a dynamic transformation of VP40 dimer into the octameric ring. Deep sequencing reveals a binding preference of the VP40 ring for the 3′ untranslated region of cellular mRNA and a guanine- and adenine-rich binding motif. Complementary analyses of the nucleic-acid-induced VP40 ring by native mass spectrometry, electron microscopy, and X-ray crystal structures at 1.8 and 1.4 Å resolution reveal the stoichiometry of RNA binding, as well as an interface involving a key guanine nucleotide. The host factor-induced structural transformation of protein structure in response to specific RNA triggers in the Ebola virus life cycle presents unique opportunities for therapeutic inhibition., Graphical abstract, In brief Landeras-Bueno et al. find that the Ebola virus matrix protein VP40 transforms to its essential octameric ring form when triggered by binding sequences in the 3′ UTR of host cell mRNA. Small RNAs/DNAs are sufficient to induce this transformation, suggesting a potential for therapeutic disruption of the virus life cycle.

Native ion mobility mass spectrometry (MS) and surface induced dissociation (SID) are applied to study the integral membrane protein complexes AmtB and AqpZ. Fragments produced from SID are consistent with the solved structures of these complexes. SID is, therefore, a promising tool for characterization of membrane protein complexes.

Among all enzymes in nature, RNase P is unique in that it can use either an RNA- or a protein-based active site for its function: catalyzing cleavage of the 5′-leader from precursor tRNAs (pre-tRNAs). The well-studied catalytic RNase P RNA uses a specificity module to recognize the pre-tRNA and a catalytic module to perform cleavage. Similarly, the recently discovered proteinaceous RNase P (PRORP) possesses two domains – pentatricopeptide repeat (PPR) and metallonuclease (NYN) – that are present in some other RNA processing factors. Here, we combined chemical modification of lysines and multiple-reaction monitoring mass spectrometry to identify putative substrate-contacting residues in Arabidopsis thaliana PRORP1 (AtPRORP1), and subsequently validated these candidate sites by site-directed mutagenesis. Using biochemical studies to characterize the wild-type (WT) and mutant derivatives, we found that AtPRORP1 exploits specific lysines strategically positioned at the tips of it's V-shaped arms, in the first PPR motif and in the NYN domain proximal to the catalytic center, to bind and cleave pre-tRNA. Our results confirm that the protein- and RNA-based forms of RNase P have distinct modules for substrate recognition and cleavage, an unanticipated parallel in their mode of action.

Herein we demonstrate the first adaptation of surface-induced dissociation in a modified high-mass range, high-resolution Orbitrap mass spectrometer. The SID device was designed to be installed in the Q-Exactive series of Orbitrap mass spectrometers with minimal disruption of standard functions. The performance of the SID-Orbitrap instrument has been demonstrated with several protein complex and ligand-bound protein complex systems ranging from 53 to 336 kDa. We also address the effect of ion source temperature on native protein-ligand complex ions as assessed by SID. Results are consistent with previous findings on quadrupole time-of-flight instruments and suggest that SID coupled to high-resolution MS is well-suited to provide information on the interface interactions within protein complexes and ligand-bound protein complexes.

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.

Surface-induced dissociation (SID) is a powerful means of deciphering protein complex quaternary structures due to its capability of yielding dissociation products that reflect the native structures of protein complexes in solution. Here we explore the suitability of SID to locate the ligand binding sites in protein complexes. We studied C-reactive protein (CRP) pentamer, which contains a ligand binding site within each subunit, and cholera toxin B (CTB) pentamer, which contains a ligand binding site between each adjacent subunit. SID dissects ligand-bound CRP into subcomplexes with each subunit carrying predominantly one ligand. In contrast, SID of ligand-bound CTB results in the generation of subcomplexes with a ligand distribution reflective of two subunits contributing to each ligand binding site. SID thus has potential application in localizing sites of small ligand binding for multisubunit protein–ligand complexes.

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids onto tRNAs. To avoid mistranslation, editing mechanisms evolved to maintain tRNA aminoacylation fidelity. For instance, while rejecting the majority of non-cognate amino acids via discrimination in the synthetic active site, prolyl-tRNA synthetase (ProRS) misactivates and mischarges Ala and Cys, which are similar in size to cognate Pro. Ala-tRNAPro is specifically hydrolyzed by the editing domain of ProRS in cis, while YbaK, a free-standing editing domain, clears Cys-tRNAPro in trans. ProXp-ala is another editing domain that clears Ala-tRNAPro in trans. YbaK does not appear to possess tRNA specificity, readily deacylating Cys-tRNACysin vitro. We hypothesize that YbaK binds to ProRS to gain specificity for Cys-tRNAPro and avoid deacylation of Cys-tRNACys in the cell. Here, in vivo evidence for ProRS-YbaK interaction was obtained using a split-green fluorescent protein assay. Analytical ultracentrifugation and native mass spectrometry were used to investigate binary and ternary complex formation between ProRS, YbaK, and tRNAPro. Our combined results support the hypothesis that the specificity of YbaK toward Cys-tRNAPro is determined by the formation of a three-component complex with ProRS and tRNAPro and establish the stoichiometry of a ‘triple-sieve’ editing complex for the first time.

SUMMARYThe exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. We show that EJC originates as an SR-rich mega-dalton sized RNP that contains RNPS1 but lacks CASC3. After mRNP export to the cytoplasm and before translation, the EJC undergoes a remarkable compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general whereas CASC3 is needed for NMD of only select mRNAs. The promotion of switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD.

Salmonella enterica elicits intestinal inflammation to gain access to nutrients. One of these nutrients is fructose-asparagine (F-Asn). The availability of F-Asn to Salmonella during infection is dependent upon Salmonella pathogenicity islands 1 and 2, which in turn are required to provoke inflammation. Here, we determined that F-Asn is present in mouse chow at approximately 400 pmol/mg (dry weight). F-Asn is also present in the intestinal tract of germfree mice at 2,700 pmol/mg (dry weight) and in the intestinal tract of conventional mice at 9 to 28 pmol/mg. These findings suggest that the mouse intestinal microbiota consumes F-Asn. We utilized heavy-labeled precursors of F-Asn to monitor its formation in the intestine, in the presence or absence of inflammation, and none was observed. Finally, we determined that some members of the class Clostridia encode F-Asn utilization pathways and that they are eliminated from highly inflamed Salmonella -infected mice. Collectively, our studies identify the source of F-Asn as the diet and that Salmonella -mediated inflammation is required to eliminate competitors and allow the pathogen nearly exclusive access to this nutrient.

Salmonella enterica serovar Typhimurium is the only organism demonstrated to utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. In this report, we first used a bioinformatics approach to identify other microorganisms that encode homologs of the Salmonella F-Asn utilization enzymes FraB (deglycase), FraD (kinase), and FraE (asparaginase). These candidate organisms were then tested with up to four different methods to confirm their ability to utilize F-Asn. The easiest and most broadly applicable method utilized a biological toxicity assay, which is based on the observation that F-Asn is toxic to a Salmonella fraB mutant. Candidate organisms were grown in a rich medium containing F-Asn, and depletion of F-Asn from the medium was inferred by the growth of a Salmonella fraB mutant in that same medium. For select organisms, the toxicity assay was cross-validated by direct mass spectrometry-aided measurement of F-Asn in the spent-culture media and through demonstration of FraB and FraD enzyme activity in cellular extracts. For prototrophs, F-Asn utilization was additionally confirmed by growth in a minimal medium containing F-Asn as the sole carbon source. Collectively, these studies established that Clostridium bolteae , Clostridium acetobutylicum , and Clostridium clostridioforme can utilize F-Asn, but Clostridium difficile cannot; Klebsiella oxytoca and some Klebsiella pneumoniae subspecies can utilize F-Asn; and some Citrobacter rodentium and Citrobacter freundii strains can also utilize F-Asn. Within Salmonella enterica , the host-adapted serovars Typhi and Paratyphi A have lost the ability to utilize F-Asn. IMPORTANCE Fructose-asparagine (F-Asn) is a precursor to acrylamide that is found in human foods, and it is also a nutrient source for Salmonella enterica , a foodborne pathogen. Here, we determined that among the normal intestinal microbiota, there are species of Clostridium that encode the enzymes required for F-Asn utilization. Using complementary experimental approaches, we have confirmed that three members of Clostridium , two members of Klebsiella , and two members of Citrobacter can indeed utilize F-Asn. The Clostridium spp. likely compete with Salmonella for F-Asn in the gut and contribute to competitive exclusion. FraB, one of the enzymes in the F-Asn utilization pathway, is a potential drug target because inhibition of this enzyme leads to the accumulation of a toxic metabolite that inhibits the growth of Salmonella species. This study identifies the potential off-target organisms that need to be considered when developing therapeutics directed at FraB.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

This communication reports the identification of gas phase isomers in monolayer-protected silver clusters. Two different isomers of Ag11(SG)7(-) (SG-gulathione thiolate) with different drift times have been detected using combined electrospray ionization (ESI) and ion mobility (IM) mass spectrometry (MS). Surface induced dissociation (SID) of the 3(-) charge state of such clusters shows charge stripping to give the 1(-) charged ion with some sodium attachment, in addition to fragmentation. SID and collision induced dissociation (CID) for Ag11(SG)7(-) suggest different pathways being accessed with each method. SID was introduced for the first time for the study of monolayer-protected clusters.

Pseudomonas aeruginosa is an important bacterial opportunistic pathogen, presenting a significant threat towards individuals with underlying diseases such as cystic fibrosis. The transcription factor AmrZ regulates expression of multiple P. aeruginosa virulence factors. AmrZ belongs to the ribbon-helix-helix protein superfamily, in which many members function as dimers, yet others form higher order oligomers. In this study, four independent approaches were undertaken and demonstrated that the primary AmrZ form in solution is tetrameric. Deletion of the AmrZ C-terminal domain leads to loss of tetramerization and reduced DNA binding to both activated and repressed target promoters. Additionally, the C-terminal domain is essential for efficient AmrZ-mediated activation and repression of its targets.

Unique fragmentation of the IMS-resolved conformers of triply charged bradykinin is shown to occur at very low collision energies by quadrupole CID. For the major C conformation of bradykinin, the unique fragmentation appears to result from a subpopulation that has a different average CCS than the dominant C population that has been previously identified as TTC (trans, trans, cis at the three Pro residues). The three major gas-phase isomers exhibit minimal differences in fragmentation at collision energies greater than 12 eV, presumably because the amide bonds of the proline residues isomerize prior to dissociation at these higher energies. The differences in fragmentation observed in the low collision energy regime (

The cooperativity of ligand binding is central to biological regulation and new approaches are needed to quantify these allosteric relationships. Herein, we exploit a suite of mass spectrometry (MS) experiments to provide novel insights into homotropic Cu-binding cooperativity, gas- phase stabilities and conformational ensembles of the D2- symmetric, homotetrameric copper-sensitive operon repressor (CsoR) as a function of Cu I ligation state. Cu I binding is overall positively cooperative, but is characterized by distinct ligation state-specific cooperativities. Structural transitions occur upon binding the first and fourth Cu I , with the latter occurring with significantly higher cooperativity than previous steps; this results in the formation of a holo-tetramer that is markedly more resistant than apo-, and partially ligated CsoR tetramers toward surface-induced dissociation (SID). The structural, dynamic and thermodynamic origins of cooperativity of ligand binding (allostery) is a subject of considerable interest, motivated by the central role this process plays in the regulation of biological activity. (1) Cooperativity can be positive or negative and involve the binding of the same (homotropic) or different (heterotropic) ligands, often to an homooligomeric protein. (2, 3) Bacterial repressors that function to control transition metal bioavail- ability in cells are typically homodimeric or homotetrameric and minimally bind two "ligands": a DNA operator found upstream of metal-regulated genes and a specific (cognate) transition metal ion(s). (4) Although a robust thermodynamic framework capable of quantifying both homo- and hetero- tropic cooperativity in these systems is available, (5) these ensemble-based methods suffer from the limitation that a specific, partially ligated state can not be studied independ- ently of other states. Such step-wise insights are required to understand allosteric coupling beyond a generally phenom- enological description. (1)

Ticks are vectors for disease transmission because they are indiscriminant in their feeding on multiple vertebrate hosts, transmitting pathogens between their hosts. Identifying the hosts on which ticks have fed is important for disease prevention and intervention. We have previously shown that hemoglobin (Hb) remnants from a host on which a tick fed can be used to reveal the host's identity. For the present research, blood was collected from 33 bird species that are common in the U.S. as hosts for ticks but that have unknown Hb sequences. A top-down-assisted bottom-up mass spectrometry approach with a customized searching database, based on variability in known bird hemoglobin sequences, has been devised to facilitate fast and complete sequencing of hemoglobin from birds with unknown sequences. These hemoglobin sequences will be added to a hemoglobin database and used for tick host identification. The general approach has the potential to sequence any set of homologous proteins completely in a rapid manner. Graphical Abstract ᅟ.

Inorganic metal oxides have shown potential as matrices for assisting in laser desorption ionization with advantages over the aromatic acids typically used. Rhenium and tungsten oxides are attractive options due to their high work functions and relative chemical inertness. In this work, it is shown that ReO3 and WO3 , in microparticle (μP) powder forms, can efficiently facilitate ionization of various types of small molecules and provide minimized background contamination at analyte concentrations below 1 ng/µL. This study shows that untreated inorganic WO3 and ReO3 particles are valid matrix options for detection of protonatable, radical, and precharged species under laser desorption ionization. Qualitatively, the WO3 μP showed improved detection of apigenin, sodiated glucose, and precharged analyte choline, while the ReO3 μP allowed better detection of protonated cocaine, quinuclidine, ametryn, and radical ions of polyaromatic hydrocarbons at detection levels as low as 50 pg/µL. For thermometer ion survival yield experiments, it was also shown that the ReO3 powder was significantly softer than α-cyano-4-hydroxycinnaminic acid. Furthermore, it provided higher intensities of cocaine and polyaromatic hydrocarbons, at laser flux values equal to those used with α-cyano-4-hydroxycinnaminic acid.

SummaryUnderstanding of protein complex assembly and the effect of ligand binding on their native topologies is integral to discerning how alterations in their architecture can affect function. Probing the disassembly pathway may offer insight into the mechanisms through which various subunits self-assemble into complexes. Here, a gas-phase dissociation method, surface-induced dissociation (SID) coupled with ion mobility (IM), was utilized to determine whether disassembly pathways are consistent with the assembly of three homotetramers and to probe the effects of ligand binding on conformational flexibility and tetramer stability. The results indicate that the smaller interface in the complex is initially cleaved upon dissociation, conserving the larger interface, and suggest that assembly of a D2 homotetramer from its constituent monomers occurs via a C2 dimer intermediate. In addition, we demonstrate that ligand-mediated changes in tetramer SID dissociation behavior are dependent on where and how the ligand binds.

Computational protein design provides the tools to expand the diversity of protein complexes beyond those found in nature. Understanding the rules that drive proteins to interact with each other enables the design of protein–protein interactions to generate specific protein assemblies. In this work, we designed protein–protein interfaces between dimers and trimers to generate dodecameric protein assemblies with dihedral point group symmetry. We subsequently analyzed the designed protein complexes by native MS. We show that the use of ion mobility MS in combination with surface-induced dissociation (SID) allows for the rapid determination of the stoichiometry and topology of designed complexes. The information collected along with the speed of data acquisition and processing make SID ion mobility MS well-suited to determine key structural features of designed protein complexes, thereby circumventing the requirement for more time- and sample-consuming structural biology approaches.

The direct determination of the overall topology and inter-subunit contacts of protein complexes plays an integral role in understanding how different subunits assemble into biologically relevant multisubunit complexes. Mass spectrometry has emerged as a useful structural biological tool because of its sensitivity, high tolerance for heterogeneous mixtures and the fact that crystals are not required. Perturbation of subunit interfaces in solution followed by gas-phase detection using mass spectrometry is a current means of probing the disassembly and hence assembly of protein complexes. Herein, we present an alternative method that employs native mass spectrometry coupled with ion mobility and two stages of surface induced dissociation (SID) where protein complexes are dissociated into subcomplexes in the first SID stage. The subcomplexes are then separated by ion mobility and subsequently fragmented into their individual monomers in the second SID stage (SID-IM-SID), providing information on how individual subunits assemble into protein complexes with different native topologies. The results also illustrate complex dependent differences in charge redistribution onto individual monomers obtained in SID-IM-SID.

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein-protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein-protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein-protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein-protein interactions.

Infrared multiple-photon dissociation (IRMPD) action spectroscopy was performed on the b2 + fragment ion from the protonated PPG tripeptide. Comparison of the experimental infrared spectrum with computed spectra for both oxazolone and diketopiperazine structures indicates that the majority of the fragment ion population has an oxazolone structure with the remainder having a diketopiperazine structure. This result is in contrast with a recent study of the IRMPD action spectrum of the PP b2 + fragment ion from PPP, which was found to be nearly 100% diketopiperazine (Martens et al. Int. J. Mass Spectrom. 2015, 377, 179). The diketopiperazine b2 + ion is thermodynamically more stable than the oxazolone but normally requires a trans/cis peptide bond isomerization in the dissociating peptide. Martens et al. showed through IRMPD action spectroscopy that the PPP precursor ion was in a conformation in which the first peptide bond is already in the cis conformation and thus it was energetically favorable to form the thermodynamically-favored diketopiperazine b2 + ion. In the present case, solution-phase NMR spectroscopy and gas-phase IRMPD action spectroscopy show that the PPGprecursor ion has its first amide bond in a trans configuration suggesting that the third residue is playing an important role in both the structure of the peptide and the associated ring-closure barriers for oxazolone and diketopiperazine formation.

The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework, IsoLAIT (Isotope-Labeled, Activity-based Identification and Tracking), designed for bisubstrate systems, the common substrate, such as S-adenosyl-L-methionine (AdoMet) for methyltransferases, is replaced by an analogue (e.g., S-adenosyl-L-vinthionine) that, as a probe, creates a tightly bound [enzyme•substrate-probe] complex upon catalysis by thiopurine-S-methyltransferase (TPMT, EC 2.1.1.67). Then, this persistent complex is identified by native mass spectrometry from the cellular milieu without separation. Furthermore, the probe’s isotope pattern flags even unknown substrates and enzymes. IsoLAIT can be broadly applicable for other enzyme systems, particularly those catalyzing group transfer and with multiple substrates, such as glycosyltransferases and kinases.

SummaryDefensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front-line immune defense against various pathogens. In addition to exerting direct antibacterial activities, defensins inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been proposed to explain defensins’ enigmatic efficiency toward various toxins. In this study, we showed that binding of neutrophil α-defensin HNP1 to affected bacterial toxins caused their local unfolding, potentiated their thermal melting and precipitation, exposed new regions for proteolysis, and increased susceptibility to collisional quenchers without causing similar effects on tested mammalian structural and enzymatic proteins. Enteric α-defensin HD5 and β-defensin hBD2 shared similar toxin-unfolding effects with HNP1, albeit to different degrees. We propose that protein susceptibility to inactivation by defensins is contingent to their thermolability and conformational plasticity and that defensin-induced unfolding is a key element in the general mechanism of toxin inactivation by human defensins.

We demonstrate that surface-induced dissociation (SID) coupled with ion mobility-mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry and quaternary structure of a multi-subunit ribonucleoprotein (RNP) complex assembled in Mg2+. We investigated here Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step-wise, Mg2+-dependent reconstitutions of Pfu RNase P using its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21•RPP29 and POP5•RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins alone showed RPP21•RPP29 and (POP5•RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly.

Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer.

Soluble guanylate cyclase (sGC) plays a central role in the cardiovascular system and is a drug target for the treatment of pulmonary hypertension. While the three-dimensional structure of sGC is unknown, studies suggest that binding of the regulatory domain to the catalytic domain maintains sGC in an autoinhibited basal state. The activation signal, binding of NO to heme, is thought to be transmitted via the regulatory and dimerization domains to the cyclase domain and unleashes the full catalytic potential of sGC. Consequently, isolated catalytic domains should show catalytic turnover comparable to that of activated sGC. Using X-ray crystallography, activity measurements, and native mass spectrometry, we show unambiguously that human isolated catalytic domains are much less active than basal sGC, while still forming heterodimers. We identified key structural elements regulating the dimer interface and propose a novel role for residues located in an interfacial flap and a hydrogen bond network as key modulators of the orientation of the catalytic subunits. We demonstrate that even in the absence of the regulatory domain, additional sGC domains are required to guide the appropriate conformation of the catalytic subunits associated with high activity. Our data support a novel regulatory mechanism whereby sGC activity is tuned by distinct domain interactions that either promote or inhibit catalytic activity. These results further our understanding of heterodimerization and activation of sGC and open additional drug discovery routes for targeting the NO–sGC–cGMP pathway via the design of small molecules that promote a productive conformation of the catalytic subunits or disrupt inhibitory domain interactions.

Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-responsive unnatural amino acids phenylalanine-4'-azobenzene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and methyl-o-nitropiperonyllysine (mNPK) at strategic positions of HisF. The light-mediated isomerization of AzoF at position 55 (fS55AzoF(E) fS55AzoF(Z)) resulted in a reversible 10-fold regulation of HisH activity. The light-mediated decaging of NPY at position 39 (fY39NPY -> fY39) and of mNPK at position 99 (fK99mNPK -> fK99) led to a 4- to 6-fold increase of HisH activity. Molecular dynamics simulations explained how the unnatural amino acids interfere with the allosteric machinery of ImGPS and revealed additional aspects of HisH stimulation in wild-type ImGPS. Our findings show that unnatural amino acids can be used as a powerful tool for the spatio-temporal control of a central metabolic enzyme complex by light.

LKB1 is functionally lost in 30% of lung adenocarcinoma cases. There are no targeted therapies available for LKB1-loss tumors. Previous studies of LKB1-loss in a mouse model of pancreatic cancer suggested that LKB1 loss drives DNA hypermethylation and serine biogenesis. DNA methyltransferases (DNMTs) maintain CpG methylation, which suppresses gene transcription. The primary substrate for DNMTs is S-adenosyl Methionine (SAMe), and the maintenance of CpG methylation has been shown to be dependent on continued regeneration of SAMe. KRAS status is associated both with LKB1 loss and with a CpG island hypermethylation phenotype (CIMP). Using a previously published 16-gene signature for LKB1 loss, we set out to confirm whether the changes that occur in the mouse model of pancreatic cancer also occur in lung cancer. We applied our signature to samples in The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset (signature-positive samples are described as “LKB1-loss”), which allowed us to compare both gene expression data as well as methylation data from Illumina 450k microarrays. We then developed a cell-line model for LKB1-loss in order to study the mechanism by which LKB1 regulates DNA methylation. We observed that LKB1-loss is associated with widespread demethylation of the genome in TCGA samples. Approximately 14,000 CpG sites were significantly hypomethylated in LKB1-loss tumors, while only 200 were hypermethylated (adjusted p-value cutoff = 0.05). Furthermore, Student’s t-test showed that LKB1-loss samples have significantly lower average methylation (β-values), regardless of KRAS status. Using motif analysis, we found that demethylated CpG sites are significantly enriched for FOXA1/2/3 consensus binding sites. FOXA transcription factors are well known as pioneering transcription factors that mediate demethylation. We discovered in vitro that FOXA1 localization and turnover is dependent on LKB1 expression, specifically the activity of downstream kinase SIK. Rather than showing enhanced serine biogenesis, LKB1-loss tumors express less DNMT1 and LC-MS/MS analysis shows depletion of SAMe. Finally, in vitro models of LKB1-loss show an increased sensitivity to DNMT inhibitors and FOXA1 silencing. Together, these observations suggest that LKB1-loss lung adenocarcinoma patients have a distinct hypomethylation profile. This profile may make them uniquely sensitive to DNMT-inhibition in combination with other therapies, a hypothesis which we will test in future studies. Citation Format: Michael J. Koenig, Joseph Amann, Bernice Agana, Jacob Kaufman, Vicki Wysocki, Christopher Oakes, David P. Carbone. LKB1 loss in lung adenocarcinoma leads to global hypomethylation and altered FOXA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 824.

Paper spray (PS) ionization, an ambient ionization method, has previously been explored as a direct and fast method for mass spectrometric analysis of complex mixtures. It has been applied to the analysis of a wide variety of compounds, mostly small molecules. The work reported here extends the application of PS ionization to noncovalent protein complexes on an ion mobility tandem mass spectrometer. Similar mass spectra for protein complexes were obtained by PS ionization and nanoflow electrospray ionization (nESI), indicating that intact protein complexes can be preserved in PS ionization. In addition, collisional cross sections measured by ion mobility provide evidence that the protein assemblies may remain compact by PS ionization. With PS, it is possible to detect hemoglobin tetramer from a blood sample with minimal sample preparation. This is the first report to show that PS ionization is a promising ionization method for nonconvalent protein complexes.

Ixodes scapularis Say, 1821 larvae were fed on mice and allowed to molt under laboratory conditions. A liquid chromatography—tandem mass spectrometry-based proteomic study was conducted to identify the type of mammalian proteins present in the derived nymphal ticks at different time intervals after molting. Albumin was present for 85 d; transferrin was present for 29 d; and, more importantly, hemoglobin remained detectable for up to 309 d postmolting. Peptides of actin, keratin, and tubulin are highly similar between mouse and tick, and therefore, unambiguous assignment of these proteins to different species was not possible. Establishing a time line for the persistence of hemoglobin, one of the most abundant blood proteins, at detectable levels in ticks after the bloodmeal and molting advances our efforts to use this protein to identify the host species.

Tandem mass spectrometry is a tool to dissect noncovalent protein complexes into smaller substructures for quaternary structure analysis. The commonly used activation method, collision induced dissociation (CID), often provides limited structural information from the typical dissociation pattern where unfolded monomers are ejected from the protein complex. In contrast, surface-induced dissociation (SID) has been shown to be very effective at dissociating protein complexes with less unfolding than CID. We present here SID of a large noncovalent tetradecamer protein, GroEL (801 kDa). A wide variety of products, including heptamers representative of the native topology, are released from the precursor upon SID, significantly different from the ubiquitous monomer ejection in CID. Enhanced dissociation into heptamers is observed when the charge states of the GroEL precursor are reduced by adding triethylammonium acetate into the spraying buffer. Ion mobility is utilized after SID to separate products overlapping in m/z to simplify the SID spectra. Compact heptamers from the charge-reduced tetradecamer are clearly distinguished from other overlapping species. SID can be very useful for quaternary structure studies of large noncovalent protein complexes, as manifested by the GroEL data where the tetradecamer dissociates into heptamers, reflecting the native topology of the complex.

SgrAI is a type IIF restriction endonuclease that cuts an unusually long recognition sequence and exhibits self-modulation of DNA cleavage activity and sequence specificity. Previous studies have shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of the SgrAI-DNA complex as well as the basic building block of the oligomers have not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI-DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD) with one SgrAI dimer bound to two precleaved duplex DNA molecules each containing one-half of the SgrAI primary recognition sequence. The oligomers contain variable numbers of DBDs with as many as 19 DBDs. Observation of the large oligomers shows that nanoelectrospray ionization (nano-ESI) can preserve the proposed activated form of an enzyme. Finally, the collision cross section of the SgrAI-DNA oligomers measured by IM-MS was found to have a linear relationship with the number of DBDs in each oligomer, suggesting a regular, repeating structure.

Adiponectin is an adipokine with insulin-sensitizing, anti-inflammatory, and cardiac protective actions. It homo-oligomerizes into trimers, hexamers, and higher molecular weight (HMW) species, which are not fully characterized. We describe high-resolution separation of adiponectin oligomers under native conditions in polyacrylamide gel coupled with methods for producing standards to provide facile and accurate identification of the oligomers. Using these procedures, adiponectin trimers in human and rodent plasma were found to migrate as two distinct populations. Distributions of these two populations are linearly proportional in plasma from type 2 diabetic patients before (R(2)=0.903, P0.001) and after (R(2)=0.960, P0.0001) 12weeks of treatment with pioglitazone as well as from control subjects (R(2)=0.891, P0.0001). In addition, HMW adiponectin could be separated into three distinct oligomers: nonamer (9mer), dodecamer (12mer), and the previously characterized octadecamer (18mer). Plasma concentrations of all oligomers increased on pioglitazone treatment, with the largest fold increase being observed in 9mers and 12mers compared with baseline. Increasing concentrations of adiponectin during oligomerization in vitro led to a disproportionate increase in 18mers. The difference between in vivo and in vitro observations suggests that higher total adiponectin protein concentration contributes to pioglitazone's ability to enhance HMW adiponectin levels, but additional factors likely affect oligomer assembly or turnover independently.

Arc repressor is a homodimeric protein with a ribbon-helix-helix fold. A single polar-to- hydrophobic substitution (N11L) at a solvent-exposed position leads to population of an alternate dimeric fold in which 310 helices replace a b-sheet. Here we find that the variant Q9V/N11L/R13V (S-VLV), with two additional polar-to-hydrophobic surface mutations in the same b-sheet, forms a highly stable, reversibly folded octamer with approximately half thea-helical content of wild-type Arc. At low protein concentration and low ionic strength, S-VLV also populates both dimeric topologies previously observed for N11L, as judged by NMR chemical shift comparisons. Thus, accumulation of simple hydrophobic mutations in Arc progressively reduces fold specificity, leading first to a sequence with two folds and then to a manifold bridge sequence with at least three different topologies. Residues 9-14 of S-VLV form a highly hydrophobic stretch that is predicted to be amyloidogenic, but we do not observe aggregates of higher order than octamer. Increases in sequence hydrophobicity can promote amyloid aggregation but also exert broader and more complex effects on fold specificity. Altered native folds, changes in fold coupled to oligomerization, toxic pre-amyloid oligomers, and amyloid fibrils may represent a near continuum of accessible alternatives in protein structure space.

Understanding protein tertiary and quaternary structures is crucial to a better understanding of their biological functions. Here we illustrate for tryptophan synthase that tandem mass spectrometry (MS/MS) reveals not only protein subunit architectures, but also protein unfolding behavior when coupled with ion mobility (IM). In the present study, we verified the subunit arrangement with surface induced dissociation (SID). We are able to correlate experimental results by IM with those obtained in unfolding simulations for the hetero-tetramer Tryptophan Synthase (TS) protein complex by identifying the presence of at least three stable intermediates (I1, I2, and I3) during the unfolding process in collision induced dissociation (CID). We illustrate that the unfolding of the TS complex is likely due to the initial unfolding of an α-monomer subunit, followed by the unfolding of the second α-monomers. We also illustrate the ability of this combination of techniques to not only identify conformational changes of TS upon addition of D,L-α-glycerol phosphate (GP), but also to determine the location of the ligand, which is buried within the α-monomer of the TS.

Glu, Gln, Pro, and Ala are the main amino acids involved in ammonia detoxification in mosquitoes. In order to develop a tandem mass spectrometry method (MS(2)) to monitor each carbon of the above isotopically-labeled (13)C-amino acids for metabolic studies, the compositions and origins of atoms in fragments of the protonated amino acid should be first elucidated. Thus, various electrospray (ESI)-based MS(2) tools were employed to study the fragmentation of these unlabeled and isotopically-labeled amino acids and better understand their dissociation pathways. A broad range of fragments, including previously-undescribed low m/z fragments was revealed. The formulae of the fragments (from m/z 130 down to m/z 27) were confirmed by their accurate masses. The structures and conformations of the larger fragments of Glu were also explored by ion mobility mass spectrometry (IM-MS) and gas-phase hydrogen/deuterium exchange (HDX) experiments. It was found that some low m/z fragments (m/z 27-30) are common to Glu, Gln, Pro, and Ala. The origins of carbons in these small fragments are discussed and additional collision induced dissociation (CID) MS(2) fragmentation pathways are proposed for them. It was also found that small fragments (≤m/z 84) of protonated, methylated Glu, and methylated Gln are the same as those of the underivatized Glu and Gln. Taken together, the new approach of utilizing low m/z fragments can be applied to distinguish, identify, and quantify (13)C-amino acids labeled at various positions, either in the backbone or side chain.

Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The β1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

Fungal cell walls contain β-glucan polysaccharides that stimulate immune responses when recognized by host immune cells. The fungal pathogen Histoplasma capsulatum minimizes detection of β-glucan by host cells through at least two mechanisms: concealment of β-glucans beneath α-glucans and enzymatic removal of any exposed β-glucan polysaccharides by the secreted glucanase Eng1. Histoplasma yeasts also secrete the putative glucanase Exg8, which may serve a similar role as Eng1 in removing exposed β-glucans from the yeast cell surface. Here, we characterize the enzymatic specificity of the Eng1 and Exg8 proteins and show that Exg8 is an exo-β1,3-glucanase and Eng1 is an endo-β1,3-glucanase. Together, Eng1 and Exg8 account for nearly all of the total secreted glucanase activity of Histoplasma yeasts. Both Eng1 and Exg8 proteins are secreted through a conventional secretion signal and are modified post-translationally by O-linked glycosylation. Both glucanases have near maximal activity at temperature and pH conditions experienced during infection of host cells, supporting roles in Histoplasma pathogenesis. Exg8 has a higher specific activity than Eng1 for β1,3-glucans; yet despite this, Exg8 does not reduce detection of yeasts by the host β-glucan receptor Dectin-1. Exg8 is largely dispensable for virulence in vivo, in contrast to Eng1. These results show that Histoplasma yeasts secrete two β1,3-glucanases and that Eng1 endoglucanase activity is the predominant factor responsible for removal of exposed cell wall β-glucans to minimize host detection of Histoplasma yeasts.

Insertions in the Salmonella enterica fra locus, which encodes the fructose-asparagine (F-Asn) utilization pathway, are highly attenuated in mouse models of inflammation (>1000-fold competitive index). Here, we report that F-Asn is bacteriostatic to a fraB mutant (IC50 19 μM), but not to the wild-type or a fra island deletion mutant. We hypothesized that the presence of FraD kinase and absence of FraB deglycase causes build-up of a toxic metabolite: 6-phosphofructose-aspartate (6-P-F-Asp). We used biochemical assays to assess FraB and FraD activities, and mass spectrometry to confirm that the fraB mutant accumulates 6-P-F-Asp. These results, together with our finding that mutants lacking fraD or the fra island are not attenuated in mice, suggest that the extreme attenuation of a fraB mutant stems from 6-P-F-Asp toxicity. Salmonella FraB is therefore an excellent drug target, a prospect strengthened by the absence of the fra locus in most of the gut microbiota.

Nitrile hydratase metalloenzymes are unique and important biocatalysts that are used industrially to produce high value amides from their corresponding nitriles. After more than three decades since their discovery, the mechanism of this class of enzymes is becoming clear with evidence from multiple recent studies that the cysteine-derived sulfenato ligand of the active site metal serves as the nucleophile that initially attacks the nitrile. Herein we describe the first direct evidence from solution phase catalysis that the source of the product carboxamido oxygen is the protein. Using(18)O-labeled water under single turnover conditions and native high resolution protein mass spectrometry, we show that the incorporation of labeled oxygen into both product and protein is turnover-dependent and that only a single oxygen is exchanged into the protein even under multiple turnover conditions, lending significant support to proposals that the post-translationally modified sulfenato group serves as the nucleophile to initiate hydration of nitriles.

In addition, Arg, 18 O2-Arg, 15 N2-Arg and 15 N2- 18 O2-Arg were also analyzed to elucidate some of the minor fragments in greater detail. Mosquito excreta from individual females were collected before and at different times after feeding a blood meal, mixed with 15 N2-Arg, an internal standard, and then derivatized as isobutyl esters. Based on the fragmentation mechanisms of Arg standards, studied by MS 2 and MS 3 , Arg in the mosquito excreta was successfully analyzed by ESI-multiple reaction monitoring in a triple-quadrupole mass spectrometer. Arg excretion was monitored over a 120 h window before and after feeding female mosquitoes with a blood meal, with the maximum level of Arg excretion observed at 36–48 h post blood feeding. This method provides an efficient and rapid tool to quantify Arg in individual blood-fed mosquitoes, and can be applied to other organisms, whose small size severally limits the use of conventional biochemical analysis. Copyright © 2012 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article.

Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N and C termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump, CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein’s function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal, but also to play an active role in efflux.