Allosteric Regulation of Rod Photoreceptor Phosphodiesterase 6 (PDE6) Elucidated by Chemical Cross-Linking and Quantitative Mass Spectrometry

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαβγ₂) is composed of a catalytic heterodimer (Pαβ) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pβ) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαβ, Pαβ–cGMP, Pαβγ₂, and Pαβγ₂–cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαβ catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.     

A Pseudo-Atomic Model for the Capsid Shell of Bacteriophage Lambda Using Chemical Cross-Linking/Mass Spectrometry and Molecular Modeling

Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.     

Binding to Rab3A-interacting molecule RIM regulates the presynaptic recruitment of Munc13-1 and ubMunc13-2

Transmitter release at synapses between nerve cells is spatially restricted to active zones, where synaptic vesicle docking, priming, and Ca2+-dependent fusion take place in a temporally highly coordinated manner. Munc13s are essential for priming synaptic vesicles to a fusion competent state, and their specific active zone localization contributes to the active zone restriction of transmitter release and the speed of excitation-secretion coupling. However, the molecular mechanism of the active zone recruitment of Munc13s is not known. We show here that the active zone recruitment of Munc13 isoforms Munc13-1 and ubMunc13-2 is regulated by their binding to the Rab3A-interacting molecule RIM1alpha, a key determinant of long term potentiation of synaptic transmission at mossy fiber synapses in the hippocampus. We identify a single point mutation in Munc13-1 and ubMunc13-2 (I121N) that, depending on the type of assay used, strongly perturbs or abolishes RIM1alpha binding in vitro and in cultured fibroblasts, and we demonstrate that RIM1alpha binding-deficient ubMunc13-2(I121) is not efficiently recruited to synapses. Moreover, the levels of Munc13-1 and ubMunc13-2 levels are decreased in RIM1alpha-deficient brain, and Munc13-1 is not properly enriched at active zones of mossy fiber terminals of the mouse hippocampus if RIM1alpha is absent. We conclude that one function of the Munc13/RIM1alpha interaction is the active zone recruitment of Munc13-1 and ubMunc13-2.     

Structural Architecture of the Nucleosome Remodeler ISWI Determined from Cross-Linking,Mass Spectrometry,SAXS, and Modeling

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Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG₁ and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution.Antibodies and variants thereof constitute the fastest growing category of therapeutic agents, and currently more than 30 immunoglobulins (Igs)¹ have been approved for the treatment of cancer, immunological diseases, and infectious diseases (1). The success of therapeutic monoclonal antibodies (mAbs) is based on the ability to specifically target diverse antigens and activate immunological effector responses. An Ig is a “dimer of a dimer” consisting of light chains and heavy chains in which each light chain is linked to a heavy chain and the light–heavy dimers are connected by disulfide bridges to form the intact antibody. IgG is the most prevalent Ig isotype in plasma and is the most commonly used isotype for therapeutic antibodies because of its strong ability to induce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (2). The IgG₁ subtype is a 150 kDa Y-shaped glycoprotein. Its stem and arms are referred to as the fragment crystallizable (Fc) and fragment antigen binding (Fab) regions, respectively. The Fab region is composed of a variable (V) and constant (C) domain from both the light chain and the heavy chain (V_L, C_L, V_H, C_H1). Antigen binding is achieved through three highly variable complementary determining regions in each variable domain (V_L and V_H) of the Fab region. The Fc region is composed of additional constant domains of the heavy chain (C_H2 and C_H3); it mediates antibody-dependent cellular cytotoxicity through interaction with Fcγ receptors (3, 4) and activates complement-dependent cytotoxicity through interaction with C1q (5). The Fc region also interacts with the neonatal Fc receptor (FcRn), which regulates the maintenance of antibody levels in plasma and thus the half-life of endogenous and recombinant monoclonal antibodies (6). The interaction between IgG and FcRn displays a characteristic pH dependence that is the basis for the function of FcRn in IgG recycling (7). FcRn rescues and recycles IgG from lysosomal degradation by binding with low micromolar affinity to internalized IgG in the slightly acidic late endosome of, for example, vascular endothelial cells (pH < 6.5). The IgG is rescued from intracellular degradation as the IgG–FcRn complex returns to the cell surface, where the IgG is released into circulation as FcRn binding is abolished in the neutral pH of plasma (6). FcRn-mediated IgG recycling contributes to the long catabolic half-life of endogenous and therapeutic antibodies of ∼22 days (8).The FcRn is a heterodimer of an MHC-class-I-like heavy chain and a β₂-microglobulin (β₂m) light chain. The FcRn heavy chain (α-chain) is composed of three structural domains, α₁, α₂, and α₃, followed by a transmembrane region and a cytoplasmic domain. The three-dimensional structure of FcRn is similar to that of MHC class I molecules in which domains α₁ and α₂ are stacked against domain α₃ and β₂m (9, 10). The pH dependence of the IgG–FcRn interaction is attributed to highly conserved residues in both FcRn and IgG (10). The first crystal structures of rat FcRn and rat Fc revealed that FcRn binds to the C_H2 and C_H3 domains of the IgG Fc region—specifically, C_H2 residues 252–254 and 309–311, as well as C_H3 residues 434–436 (11, 12). Several positively charged histidines in the IgG C_H2 and C_H3 domains (H310, H433, H435, and H436; the latter is not found in humans) interact with acidic residues E117, E132, W133, E135, and D137 in the FcRn α₂ domain, accounting for the pH-sensitive nature of the IgG–FcRn interaction. The interface is also composed of a hydrophobic core around Fc I253 that interacts with FcRn W133 and the N-terminal I1 residue of the β₂m, which has been proposed to contact Fc residues 309–311. The interaction of FcRn and IgG occurs in a 2:1 stoichiometry, where two FcRn molecules bind to one IgG through binding sites on each heavy chain (12). Two distinct binding modes have been suggested in which the FcRn molecules bind in a symmetric or asymmetric fashion to the Fc. In symmetric models FcRns bind to opposite sites on the Fc, whereas in the asymmetric models two FcRn molecules form a homodimer with only one FcRn molecule binding the Fc directly (6, 11). The extracellular domains of rat and human FcRn have 68% sequence identity and are structurally similar (9, 10). The first crystal structure of human FcRn in complex with an engineered human Fc fragment (Fc-YTE) as well as human serum albumin was published recently (13) and showed a binding mode similar to that of rodent IgG–FcRn variants, with the exception of the additional interaction sites caused by substitutions in the Fc domain. To the best of our knowledge, no crystal structures of full-length human IgG and human FcRn are currently available.From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize the pharmacokinetics and thus ultimately the efficacy of therapeutic monoclonal antibodies. The goal of FcRn modulation is typically prolongation of the in vivo half-life in order to reduce dosing frequency and ultimately the cost of treatment. However, a shorter half-life can also be desirable, for example, for antibody–toxin conjugates or antibodies used in bioimaging (6). Several engineered therapeutic mAb variants with improved in vitro FcRn binding affinity and extended in vivo half-life have been generated via mutation of residues in the Fc domain (14–19). For example, the engineered variants of palivizumab (M252Y/S254T/T256E) (15, 16) and bevacizumab (M428L/N434S) (17) show 10- and 11-fold increases in relative FcRn affinity that result in increases of the in vivo half-life in cynomolgus monkeys of 4- and 3-fold, respectively. Mutation can also impact half-life negatively: mAb engineering can improve FcRn affinity at both pH 6 and 7.5 such that the pH-dependent release of IgGs is prohibited, leading to increased IgG clearance (16). Interestingly, post-translational modifications such as oxidation of conserved methionines in the C_H2 and C_H3 domains of IgG₁ and IgG₂ have been shown to affect FcRn affinity negatively. Antibody oxidation that can occur during production or storage significantly reduces FcRn binding in vitro (20, 21), which also translates to a reduced in vivo half-life in human FcRn transgenic mice models (22). The molecular origins of the effect of post-translational modifications on the IgG–FcRn interaction are, however, unclear. Further, the impact of FcRn binding on the conformational properties and dynamics of IgG in solution is currently not well understood.In this study we investigated the interaction between human FcRn and two variants of a full-length IgG₁ by means of hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS). HDX-MS has become a popular approach for studying protein dynamics and interactions (23–27), as the technique provides access to proteins at native solution conditions with modest sample requirements. Amide HDX rates in native proteins are highly influenced by higher order structure: fully solvated (non-hydrogen-bonded) amides exchange rapidly, whereas structurally protected (hydrogen-bonded) amides exchange up to 7 orders of magnitude slower (28, 29). Protein interactions can be studied and mapped via HDX-MS, as binding events can perturb HDX rates as solvation and hydrogen bonding changes directly in the binding interface or indirectly in conformationally linked regions. The structural resolution of a classic peptide-level HDX-MS experiment is dependent on the generation of overlapping peptides by acid-stable proteases, such as pepsin, typically used in HDX-MS workflows. More recently, the use of gas-phase fragmentation of deuterated peptides with ETD (30–33) has become a viable option for sublocalizing deuterium uptake to short peptide stretches or even individual amino acids, thus increasing the spatial resolution of the classical bottom-up HDX-MS method.Here, we used HDX-MS to probe the solution-phase interactions of human FcRn with a full-length recombinant human IgG₁ and its deglycosylated variant. Our results allowed us to map antibody and FcRn regions that displayed changes in HDX upon complex formation and examine the impact of antibody glycosylation on FcRn binding. Additionally, by coupling ETD to the HDX-MS workflow in a targeted manner, we obtained high-resolution information on the HDX of individual sites that became protected upon IgG₁–FcRn complex formation.     

Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking,Co-Immunoprecipitation and Mass Spectrometry

Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners.     

Unique Structural Changes in Calcium-Bound Calmodulin Upon Interaction with Protein 4.1R FERM Domain: Novel Insights into the Calcium-dependent Regulation of 4.1R FERM Domain Binding to Membrane Proteins by Calmodulin

Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca²⁺ but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca²⁺-dependent manner. We have previously mapped a Ca²⁺-independent CaM-binding site, pep11 (A²⁶⁴KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca²⁺ (Ca²⁺/CaM), interacts simultaneously with pep11 and with Ser¹⁸⁵ in A¹⁸¹KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca²⁺/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca²⁺/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca²⁺. Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca²⁺. Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca²⁺/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca²⁺/CaM-dependent regulation of R30 binding to membrane proteins.     

Reassessment of the Unique Mode of Binding between Angiotensin II Type 1 Receptor and Their Blockers

While the molecular structures of angiotensin II (Ang II) type 1 (AT₁) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT₁ receptor, different positions of the AT₁ receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT₁ receptor, Tyr¹¹³, Tyr¹⁸⁴, Lys¹⁹⁹, His²⁵⁶ and Gln²⁵⁷ using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT₁ receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr¹¹³ in the AT₁ receptor and the hydroxyl group of olmesartan, between Lys¹⁹⁹ and carboxyl or tetrazole groups, and between His²⁵⁶ or Gln²⁵⁷ and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys¹⁹⁹, His²⁵⁶ and Gln²⁵⁷ of AT₁ receptor. Lys¹⁹⁹ in the AT₁ receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys¹⁹⁹. The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr¹¹³. On the other hand, the n-butyl group of irbesartan may bind to Tyr¹¹³. In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT₁ receptor and for the formation of unique modes of binding.     

Electrospray Ionization-Mass Spectrometry and Tandem Mass Spectrometry Reveal Self-Association and Metal-Ion Binding of Hydrophobic Peptides: A Study of the Gramicidin Dimer

Gramicidin is a membrane pentadecapeptide that acts as a channel, allowing the passage of monovalent metal ions and assisting in bacterial cell death. The active form is a noncovalently bound dimer. One means to study the self-assembly of this peptide has been to compare the state of the peptide in various solvents ranging from hydrophilic (e.g., trifluoroethanol) to hydrophobic (e.g., n-propanol). In this article, we report the use of electrospray mass spectrometry to study the self-association of gramicidin in various organic and mixed solvents that are introduced directly into the mass spectrometer. The dimer (both homo and hetero) can survive the introduction into the gas phase, and the amount in the gas phase increases with the decreasing dielectric constant of the solvent, reflecting solution-phase behavior. Tandem mass spectrometry data reveal that the stability of dimer in the gas phase decreases with increasing metal ion size, strongly suggesting that the metal ion binds inside the dimer between the monomers.     

Characterization of the Raptor/4E-BP1 Interaction by Chemical Cross-linking Coupled with Mass Spectrometry Analysis

mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56–72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.     

Specificity and Commonality of the Phosphoinositide-Binding Proteome Analyzed by Quantitative Mass Spectrometry

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Characterization of a New Abscisic Acid-conjugated Metabolite in Crude Plant Extract by Desorption Chemical Ionization and Secondary Ion Mass Spectrometry

A new conjugate of hydroxyabscisic acid, tentatively named MeHMG-HOABA, along with a known conjugate, β-hydroxy-/?-methylglutarylhydroxyabscisic acid (HMG-HOABA), were iso-lated from immature seeds of Robinia pseudacacia and determined. Evidence for the occurrence of MeHMG-HOABA as a natural metabolite, and not as an artifact, was provided by desorption chemical ionization (DCI) and secondary ion mass spectrometry (SIMS) in conjunction with the technique of linked scanning at constant B/E. The mass spectrometric technique allows the detection and characterization of the conjugates to be analysed even in a crude plant extract.     

Analysis of Acidic Metabolites of Biogenic Amines in Bovine Retina and Vitreous and Aqueous Humour by Gas Chromatography-Negative Ion Chemical Ionisation Mass Spectrometry

Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.     

Interannual Changes in Biomass Affect the Spatial Aggregations of Anchovy and Sardine as Evidenced by Geostatistical and Spatial Indicators

Geostatistical techniques were applied and a series of spatial indicators were calculated (occupation, aggregation, location, dispersion, spatial autocorrelation and overlap) to characterize the spatial distributions of European anchovy and sardine during summer. Two ecosystems were compared for this purpose, both located in the Mediterranean Sea: the Strait of Sicily (upwelling area) and the North Aegean Sea (continental shelf area, influenced by freshwater). Although the biomass of anchovy and sardine presented high interannual variability in both areas, the location of the centres of gravity and the main spatial patches of their populations were very similar between years. The size of the patches representing the dominant part of the abundance (80%) was mostly ecosystem- and species-specific. Occupation (area of presence) appears to be shaped by the extent of suitable habitats in each ecosystem whereas aggregation patterns (how the populations are distributed within the area of presence) were species-specific and related to levels of population biomass. In the upwelling area, both species showed consistently higher occupation values compared to the continental shelf area. Certain characteristics of the spatial distribution of sardine (e.g. spreading area, overlapping with anchovy) differed substantially between the two ecosystems. Principal component analysis of geostatistical and spatial indicators revealed that biomass was significantly related to a suite of, rather than single, spatial indicators. At the spatial scale of our study, strong correlations emerged between biomass and the first principal component axis with highly positive loadings for occupation, aggregation and patchiness, independently of species and ecosystem. Overlapping between anchovy and sardine increased with the increase of sardine biomass but decreased with the increase of anchovy. This contrasting pattern was attributed to the location of the respective major patches combined with the specific occupation patterns of the two species. The potential use of spatial indices as auxiliary stock monitoring indicators is discussed.     

CNBr/Formic Acid Reactions of Methionine- and Trifluoromethionine-Containing Lambda Lysozyme: Probing Chemical and Positional Reactivity and Formylation Side Reactions by Mass Spectrometry

The cyanogen bromide (CNBr)/formic acid cleavage reactions of wild-type and trifluoromethionine (TFM)-containing recombinant lambda lysozyme were studied utilizing ESI and MALDI mass spectrometry. Detailed analysis of the mass spectra of reverse-phase HPLC-purified cleavage fragments produced from treatment of the wild-type and labeled proteins with CNBr indicated cleavage solely of methionyl peptide bonds with no observation of cleavage at TFM. N-Acetyl-TFM was also found to be resistant to reaction with CNBr, in contrast to N-acetyl-methionine. The analysis also indicated differential reactivity among the three methionine positions in the wild-type enzyme. Additionally, formylation of intact enzyme as well as peptide fragments were observed and characterized and indicated that serine, threonine, as well as C-terminal homoserine side chains are partially formylated under standard cleavage protocols.     

Artifact-Free Quantification of Free 3-Chlorotyrosine, 3-Bromotyrosine, and 3-Nitrotyrosine in Human Plasma by Electron Capture–Negative Chemical Ionization Gas Chromatography Mass Spectrometry and Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

Halogenation and nitration of biomolecules have been proposed as key mechanisms of host defense against bacteria, fungi, and viruses. Reactive oxidants also have the potential to damage host tissue, and they have been implicated in disease. In the current studies, we describe specific, sensitive, and quantitative methods for detecting three stable markers of oxidative damage: 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Our results indicate that electron capture-negative chemical ionization-gas chromatography/mass spectrometry (EC-NCI GC/MS) is 100-fold more sensitive than liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) for analyzing authentic 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine. Using an isotopomer of tyrosine to evaluate artifactual production of the analytes during sample preparation and analysis, we found that artifact generation was negligible with either technique. However, LC-MS/MS proved cumbersome for analyzing multiple samples because it required 1.5 h of run and equilibration time per analysis. In contrast, EC-NCI GC/MS required only 5 min of run time per analysis. Using EC-NCI GC/MS, we were able to detect and quantify attomole levels of free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in human plasma. Our results indicate that EC-NCI GC/MS is a sensitive and specific method for quantifying free 3-chlorotyrosine, 3-bromotyrosine, and 3-nitrotyrosine in biological fluids in a single, rapid analysis and that it avoids generating any of the analytes ex vivo.     

Backbone Structure Confirmation and Side Chain Conformation Refinement of a Bradykinin Mimic BKM-824 by Comparing Calculated 1H, 13C and 19F Chemical Shifts with Experiment

Abstract

Calculated and experimental ¹H, ¹³C and ¹⁹F chemical shifts were compared in BKM-824, a cyclic bradykinin antagonist mimic, c[Ava¹-Igl²-Ser³-DF5F⁴-Oic⁵-Arg⁶] (Ava=5-amino- valeric acid, Igl=α-(2-indanyl)glycine, DF5F=pentafluorophenylalanine, Oic=(2S,3aS,7aS)- octahydroindole-2-carboxylic acid). The conformation of BKM-824 has been studied earlier by NMR spectroscopy (M. Miskolzie et al., J. Biomolec. Struct. Dyn. 17, 947–955 (2000)). All NMR structures have qualitatively the same backbone structure but there is considerable variation in the side chain conformations. We have carried out quantum mechanical optimization for three representative NMR structures at the B3LYP/6–31G* level, constraining the backbone dihedral angles at their NMR structure values, followed by NMR chemical shift calculations at the optimized structures with the 6–311G** basis set. There is an intramolecular hydrogen bond at Ser³ in the optimized structures.

The experimental ¹³C chemical shifts at five C^α positions as well as at the C^β, C^γ and Cδ position of Ava¹, which forms part of the backbone, are well reproduced by the calculations, confirming the NMR backbone structure. A comparison between the calculated and experimental H^β chemical shifts in Igl² shows that the dominant conformation at this residue is gauche. Changes of proton chemical shifts with the scan of the χ1 angle in DF5F⁴ suggest that χ1 ≈180°. The calculated ¹H and ¹³C chemical shifts are in good agreement with experiment at the rigid residue Oic⁵. None of the models gives accurate results for Arg⁶, presumably because of its positive charge. Our study indicates that calculated NMR shifts can be used as additional constraints in conjunction with NMR data to determine protein conformations. However, to be computationally effective, a database of chemical shifts in small peptide fragments should be precalculated.