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The αββα tryptophan synthase (TS), which is part of primary metabolism, is a paradigm for allosteric communication in multienzyme complexes. In particular, the intrinsically low catalytic activity of the α-subunit TrpA is stimulated several hundredfold through the interaction with the β-subunit TrpB1. The BX1 protein from Zea mays (zmBX1), which is part of secondary metabolism, catalyzes the same reaction as that of its homologue TrpA, but with high activity in the absence of an interaction partner. The intrinsic activity of TrpA can be significantly increased through the exchange of several active-site loop residues, which mimic the corresponding loop in zmBX1. The subsequent identification of activating amino acids in the generated "stand-alone" TrpA contributes to an understanding of allostery in TS. Moreover, findings suggest an evolutionary trajectory that describes the transition from a primary metabolic enzyme regulated by an interaction partner to a self-reliant, stand-alone, secondary metabolic enzyme.

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.

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)

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.

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.

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.

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.

The quaternary structure of proteins determines their biological function, and a majority of proteins exist as oligomers in vivo with miscellaneous architectures. Mass spectrometry (MS) can be applied to study the stoichiometry and interactions of protein complexes by molecular weight measurements under gentle instrumental conditions where noncovalent interactions are preserved in the gas phase. Recently, there have been extensive efforts to also utilize ion mobility (IM) techniques combined with MS for structural studies of noncovalent biological complexes, including virus assembly pathways, because IM provides conformational information, which is not accessible by MS, for these gas-phase ions. Experimentally measured collisional cross sections (CCSs) from IM can serve as constraints for architecture determination by molecular modeling. Additionally, tandem MS can be used to dissociate gas-phase complexes. A protein assembly would ideally dissociate into various noncovalent subcomplexes, and the topology of the original complex could be derived by piecing together all the subcomplex products. The common tandem MS method, collision induced dissociation (CID), involves activation of the complexes by collision with neutral gas atoms or molecules. Typically, CID results in an “asymmetric” dissociation into highly charged monomers and complementary (n 1)-mers (although a few exceptions have been reported), and studies have suggested that unfolding of protein complexes occurs in CID. It is therefore difficult to relate the CCS measurements of CID product ions to the complexes native structure. Tandem MS can alternatively be achieved by surface induced dissociation (SID) where the complexes collide with a surface target. Previous research in our group has shown that several protein complexes dissociate in a more “symmetric” manner with SID than by CID, and have charge distributed more proportionally to the mass. We hypothesized that dissociation might occur in the absence of gradual monomer unfolding for SID because activation by SID is a single-step, higher-energy deposition, fast process that is different from the multistep, slower CID process. SID has recently been applied to determining the quaternary structure of a heterohexameric protein with information from subunit product ions such as heterotrimers unique to SID. We present herein the first IM measurements on the SID products of several protein complexes, along with comparison to CID products, by using a modified quadrupole/IM/time-offlight (Q/IM/TOF) instrument. Briefly, the precursor ions are dissociated by CID or SID cells placed in front of the IM cell. The product ions are subsequently separated based on their size, shape, and charge under the influence of a continuous series of electrical pulses and friction with neutral gas in the IM cell. The drift times of the ions are recorded, with larger and lower-charged ions experiencing longer drift times. Experimental CCSs can be derived from the measured drift times and mass-to-charge-ratios. Theoretical CCSs can be calculated from crystal structures. Nativelike ions should have experimental CCSs similar to the crystal structure, whereas unfolded ions are expected to show larger CCS values because of an increased surface area. We first examined the remaining undissociated pentamer precursor of C-reactive protein (CRP) after activation by either CID or SID. Triethylammonium acetate (TEAA) was added in the electrospraying buffer, which has been reported as a charge reducing additive to increase the stability of protein complexes in the gas phase. Without TEAA no remaining precursor could be observed in SID, even at low acceleration voltages. The addition of TEAA did not cause any remarkable structural change of the protein as the CCSs of the precursor did not change after charge reduction. The + 18 precursor of CRP was selected and activated. Examination of precursor CCSs at various SID acceleration voltages (20–50 V) reveals that most of the CRP pentamer dissociated without extensive increase in CCS of the remaining precursor. In CID, however, the CCS of the undissociated CRP pentamer first decreased at low acceleration voltages and then increased considerably above its dissociation threshold of about 80 V. It reached a stable unfolding intermediate with CID around 100 V, where the CCS does not additionally increase with increasing CID acceleration voltages (data not shown). It is impractical to determine one acceleration voltage at which the amounts of the internal energies deposited in CID and SID are identical because of different mechanisms and complications from the physical properties of large protein complexes. Nonetheless, we show here a representative comparison between CID at 100 V (Figure 1, top right) and SID at 40 V (Figure 1, bottom [*] M. Zhou, Dr. S. Dagan, Prof. V. H. Wysocki Department of Chemistry and Biochemistry, University of Arizona 1306 E. University Blvd., Tucson, AZ (USA) E-mail: vwysocki@email.arizona.edu [] Permanent address: Israel Institute for Biological Research (IIBR) POB 19, Ness Ziona 74100 (Israel)

Female mosquitoes require a blood meal for oogenesis and receive a substantial iron load in this meal in the form of holo-transferrin and hemoglobin. During the course of digestion and absorption i...

Protein identification and peptide sequencing by tandem mass spectrometry requires knowledge of how peptides fragment in the gas phase, specifically which bonds are broken and where the charge(s) resides in the products. For many peptides, cleavage at the amide bonds dominate, producing a series of ions that are designated b and y. For other peptides, enhanced cleavage occurs at just one or two amino acid residues. Surface-induced dissociation, along with gas-phase collision-induced dissociation performed under a variety of conditions, has been used to refine the general 'mobile proton' model and to determine how and why enhanced cleavages occur at aspartic acid residues and protonated histidine residues. Enhanced cleavage at acidic residues occurs when the charge is unavailable to the peptide backbone or the acidic side-chain. The acidic H of the side-chain then serves to initiate cleavage at the amide bond immediately C-terminal to Asp (or Glu), producing an anhydride. In contrast, enhanced cleavage occurs at His when the His side-chain is protonated, turning His into a weak acid that can initiate backbone cleavage by transferring a proton to the backbone. This allows the nucleophilic nitrogen of the His side-chain to attack and form a cyclic structure that is different from the 'typical' backbone cleavage structures.

Cis and trans isomers of two dinuclear platinum complexes, [cis-{Pt(NH 3 ) 2 Cl} 2 μ-(NH 2 (CH 2 ) a NH 2 )]]NO 3 ) 2 (1,1/c,c) and [trans-{Pt(NH 3 ) 2 Cl} 2 μ-(NH 2 (CH 2 ) a NH 2 )](NO 3 ) 2 (1,1/t,t), where the diamine was 1,4-butanediamine (n = 4) or 1,6-hexanediamine (n = 6), were studied using electrospray ionization surface-induced dissociation (ESI/SID) tandem mass spectrometry (MS/MS). The same fragment ions were observed for both the cis and trans isomers of each complex (n = 4 or 6), but the relative intensities were dependent on the isomer studied. The ESI/SID data and energy-resolved mass spectra show that the position of the chloride plays a significant role in the fragmentation of these ions. Two major fragmentation pathways were detected for the complexes. The cleavage of the Pt-N bond trans to chloride was the most favorable pathway for both isomers of the complexes following the ion-surface collision. The differences in the ESI/SID spectra between the cis and trans isomers can be explained by the trans effect, namely that the Pt-N bond trans to chloride is the most labile bond.

Doubly protonated substance P and two analogs alkylated at the ninth position was studied to determine the effect of N-alkylation of the amide nitrogen on the electrospray ionization/surface-induced dissociation (ESI/SID) fragmentation pattern. Thermal decomposition experiments and ab initio calculations were also used in conjunction with the ESI/SID experiments. The increase in relative abundances of the product ions resulting from the cleavage of the amide bond at the alkylation site (relative to the corresponding cleavage for substance P) can be explained by the increased basicity of the amide nitrogen in the context of the 'mobile proton' model. The relative abundances of singly charged b ions suggest a rearrangement of the amide hydrogen located N-terminal to the bond cleaved.

The utility of surface-induced dissociation (SID) to probe the structure, energetics and fragmentation mechanisms of protonated peptides is discussed and demonstrated. High internal energy deposition provided by low-energy (eV range) ion-surface collisions yields extensive fragmentation of protonated peptides, allowing relatively uncomplicated and rapid sequence analysis of oligopeptides. SID of multiply protonated peptides is illustrated for peptides with molecular mass of up to approximately 5000 u. It is also illustrated that SID combined with electrospray ionization (ESI) provides a distinctive experimental technique to determine the energetics and mechanisms of peptide fragmentation. The relative position of ESI/SID fragmentation efficiency curves (plots of percentage fragmentation vs. laboratory collision energy) for peptides can be utilized to estimate relative energetics of peptide fragmentation and even to predict proton localization sites. The observed trends support the essential role of the mobile proton model in understanding peptide fragmentation by low-energy tandem mass spectrometry.

The so-called 'deconvolution' method has been used to determine the internal energy distribution of molecular ions of an organic compound, benzene, excited by collisions with self-assembled monolayer surfaces formed on gold. The average internal energy was found to increase linearly with the laboratory collision energy. The kinetic energyinternal energy (T-V) conversion was 17% for the octadecanethiolate monolayer and 28% for the 2(perfluoroocty1)ethanethiolate monolayer surface. The results are similar to those obtained for metal carbonyl projectiles, though they indicate somewhat higher energy conversion. In addition, excitation of the projectile ion well beyond 20 eV internal energy is observed.

Low-energy collisions of ionized acetone-d, , DMSO-d, and pyridine-d, with alkanethiolate self-assembled monolayer (SAM) surfaces prepared on gold were investigated. These experiments can be used to monitor routinely the condition of the surfaces and to determine whether a surface is acceptable for a given application. In addition to the surface-induced dissociation of the molecular ions, intense peaks corresponding to the addition of H (or D) to the ionized molecules are observed in the ioit-surface collision spectra. The relative abundance of the IM + H(D)I + reaction products was found to be dependent on several variables, including (i) the chemical composition of the monolayers (alkanethiolate, perdeuteroalkanethiolate, fluorinated alkanethiolate), (ii) the chain length of the alkanethiolate monolayers, (iii) the time for which the vapor-deposited Au surfaces are exposed to the thiol solution during surface preparation and (iv) the degree of deliberate damage of the SAM film (70 eV Ar" beam bombardment). The variation in [ M + H(D)I + abundance with induced surface changes illustrates the utility of these adduct ions for the characterization of the quality of the monolayers. The general trend observed is an increase in the I M + HI + peak with the increased presence of hydrocarbon adsorbates, which in turn is believed to be directly related to increased disorder (inherent or induced) of the monolayer film. The increase in [ M + HI + is seen with a decrease in chain length, with shorter monolayer preparation times, and with an increase in surface damage. In general, a fluorinated surface that has been intentionally damaged by an Ar" beam retains its highenergy deposition characteristics and is appropriate for use as a collision target for many types of projectiles.

We report here the spectra that result when singly and doubly charged buckminsterfullerene ions, C& and Cg, collide with self-assembled monolayer films at collison energies ranging from 125 to 320 eV. The surfaces were prepared by the spontaneous assembly of octadecanethiol (CH3(CHz),7SH), perdeuteroeicosanethiol (CD,(CDz),$H), and 2-(perfluorooctyl)-ethanethiol (CF3(CFd7CH2CHZSH) on vapor-deposited gold. The fluorinated surface causes the greatest conversion of kinetic energy into internal energy; more extensive fragmentation of doubly charged buckminsterfullerene Cg is detected in collisions with this surface than in collisions with the alkanethiolate surfaces. Recent work by several groups has shown that lowenergy collisions between polyatomic ions and surfaces can produce fragmentation useful for structural characterization of ions.'-20 This technique, introduced and developed by Cooks and coworkers, is known as surface-induced dissociation (SID) and has been reviewed recently.' It has been shown that SID can produce relatively extensive fragmentation of species including ben~ene,'.~, 14, 19a.20 polycyclic aromatic hydrocarbon^,'-^, 14, 19a and peptides;', lib. '', I& in the case of peptides, low-energy collisions with surfaces have been used to extract sequence information. Ion/surface collisions result in other phenomena as well, including chemical sputtering, charge exchange, and ionlsurface reactions. ' These phenomena can have an important influence on the products of iodsurface collisions; for, example, molecular ions of pyridine extract a hydrogen from some surfaces and fragment from an excited protonated species. ', l3 Moreover, processes such as charge exchange between the projectile and the surface can influence the product yields (through neutralization). SID is proving to be a useful tool in structural characterization problems because a significant fraction of the kinetic energy of impact is deposited into the projectile as internal energy. The fraction of kinetic energy converted to internal energy is relatively high, typically 12-15% for small molecules.' Thus, if the efficiency of energy transfer holds for larger species, SID may be a useful method for depositing relatively large amounts of energy into large or non-labile species with instruments that utilize low ion energies. A particularly intriguing feature of SID is that the efficiency of energy transfer appears to increase with projectile mass, based on a limited number of studies to date. Whetten and coworkers, for example, have estimated that as much as 35% of the kinetic energy is converted to internal energy when large alkali halide cluster ions

Low energy ion-surface collisions with organic surfaces are reviewed in terms of the ions formed as studied by mass spectrometry. Chemical manipulation of the incident ion and the surface are both of interest.

Attempts to induce remote site fragmentation by using low-energy collisional activation with a neutral target gas or with a surface (surface-induced dissociation) are reported. These data, in conjunction with knowledge of the internal energy deposition associated with these activation methods, provide information on the amount of internal energy needed to promote remote site fragmentation in selected compounds. The internal energy required for remote site fragmentation of lithiated oleyl alcohol was recently estimated to be 1.3–1.9 eV. Our experiments suggest that internal energies much greater than 2 eV are required to produce detectable remote site fragmentation in several different ions, including lithiated oleyl alcohol, n-dodecylbenzenesulfonate anion, stearylsulfate anion, stearate anion and protonated octadecylamine. The results also suggest that the internal energy required to record spectra which show remote site fragmentation is compound-dependent, i.e. strongly dependent on the nature of the ionic group.

Collision of the title ion upon a stainless steel surface at near-normal incidence leads to deposition of internal energy in a well-defined narrow distribution. The energy deposited increases with laboratory collision energy and exceeds 7 eV (average) for 100 eV collisions. The translational-to-vibrational energy transfer efficiency is 15% (assuming an infinitely massive target) at 25 eV collision energy. Comparison is made with the internal energy distributions associated with gas-phase collisional activation using both low and high ion kinetic energies. The narrowness of the distribution of internal energies, the easy access to ions excited to different extents, and the high internal energies accessible, make the ionlsurface collision process superior to gas-phase collisional activation for this system. Various methods of activation are used to characterize gas-phase ions through their dissociation reactions. In spite of their success in many ion structural problems, all of these methods suffer from one or more of the following disadvantages: (i) only a small average amount of energy is deposited,'-'' (ii) a very broad range of energies is depo~ited,~-~,~~ -'~ (iii) only a limited set of ions can be studied (e.g. only ions absorbing certain photons, or only ions having a stable neutral c~unterpart),~,' ,~~~~ (iv) the ion internal energy is not kno~n~-~,~~~,~~ and (v) variation of the ion internal energy in a controlled fashion is not straightfor~ard. ~~~~~~~~~~~~~~~ These disadvantages limit the type and number of ions which can be studied, as well as the quality and quantity of information available. We wish to report our results concerning energy deposition with a promising new ion excitation method, surface-induced dissociation (SID). It is possible to excite organic ions by allowing them to collide with a stainless steel surface at small laboratory The extent of fragmentation increases with increasing collision energy, suggesting that there is a dependence of the energy deposition upon the collision energy. We now show that a narrow energy distribution is deposited in these experiments. Furthermore, the amount of energy deposited can be systematically increased up to very high values. Thus, this method allows experiments to be conducted as a function of ion internal energies. The SID method should also be useful in calibrating other activation methods which deposit unknown or ill-defined amounts of energy. The energy transfer associated with surface collisions can be characterized by ion kinetic energy measurementsz5 or by employing a recently developed method3 for estimating the distribution of internal energies, P(E), in a population of ions. The method is based on fragment ion abundances arising from a