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The molecular identification of species of interest is an important part of an imaging mass spectrometry (IMS) experiment. The high resolution accurate mass capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) have recently been shown to facilitate the identification of proteins in matrix-assisted laser desorption/ionization (MALDI) IMS. However, these experiments are typically limited to proteins giving rise to ions of relatively low m/ z due to difficulties transmitting and measuring large molecular weight ions of low charge states. Herein we have modified the source gas manifold of a commercial MALDI FT-ICR MS to regulate the gas flow and pressure to maximize the transmission of large m/ z protein ions through the ion funnel region of the instrument. By minimizing the contribution of off-axis gas disruption to ion focusing and maximizing the effective potential wall confining the ions through pressure optimization, the signal-to-noise ratios (S/N) of most protein species were improved by roughly 1 order of magnitude compared to normal source conditions. These modifications enabled the detection of protein standards up to m/ z 24 000 and the detection of proteins from tissue up to m/ z 22 000 with good S/N, roughly doubling the mass range for which high quality protein ion images from rat brain and kidney tissue could be produced. Due to the long time-domain transients (>4 s) required to isotopically resolve high m/ z proteins, we have used these data as part of an FT-ICR IMS-microscopy data-driven image fusion workflow to produce estimated protein images with both high mass and high spatial resolutions.
The physiology of N-methyl-d-aspartate (NMDA) receptors is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino-terminal domain (ATD). Recent crystal structures of GluN1-GluN2B NMDA receptors bound to agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here we applied X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology to rat NMDA receptors to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.
Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase, verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus.
© 2015 John Wiley & Sons Ltd.
Calmodulin (CaM) is a ubiquitous EF-hand calcium sensor protein that transduces calcium signals in a wide range of signaling pathways. Structural analysis of complexes with peptides has provided valuable insights into the remarkable variety in the way in which CaM interacts with and activates its targets. Among these various targets, CaM has been shown to be an essential component of a calcium-sensing regulatory apparatus for a number of voltage-gated ion channels. NMR spectroscopy has proven to be a powerful tool for the structural characterization of CaM-peptide complexes, in particular for the study of IQ motifs, which bind CaM at the basal level of calcium in cells and thereby serve to localize CaM to its sites of action. We describe here methods for the robust expression and purification of CaM isotopically enriched for NMR analysis, as well as for the complex of CaM with a peptide derived from the IQ motif sequence of the human cardiac sodium channel Na(V)1.5. We also describe methods for NMR analysis of titrations of CaM with IQ motif peptides to determine the stoichiometry of the complex and to identify the residues at the binding interface.
The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D(9k) (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca(2+), the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca(2+)-loaded calbindin D(9k) does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca(2+) is bound to the protein. A reduction in pH in the absence of Ca(2+) increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca(2+) affinity causes a transition to the micelle-associated state also in the presence of Ca(2+) when the pH is lowered. Site-specific analysis of the data indicates that calbindin D(9k) has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca(2+)-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D(9k) in the cell is discussed, along with the broader implications for the function of the S100 protein family.
Although surfactant apoproteins are known to be mediators of innate responses, their relationship to adaptive responses has not been examined extensively. We investigated possible links between surfactant apoproteins and responses to allergens by studying alterations in surfactant apoproteins A, B, and D in a murine model of allergic pulmonary inflammation. Three murine strains (BALB/c, C57BL/6, and 129J) demonstrated increased immunostaining of surfactant apoproteins A and D in nonciliated epithelial cells of noncartilaginous airways after aerosolized challenge. In contrast, surfactant apoprotein B immunostaining was unchanged. Immunoblotting demonstrated increased surfactant A in bronchoalveolar lavage fluid after allergen sensitization and challenge. Surfactant apoprotein A and D induction required T and/or B lymphocyte responses to allergen, since the induction was absent in recombinase-activating gene-deficient mice, which lack functional lymphocytes. We conclude that increased immunoreactivity of two collectins, surfactant apoproteins A and D, occurs within the response to allergen. Our findings support a model in which surfactant apoproteins A and D are important to both innate immunity and adaptive immune responses to allergens.
Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b(5) (b(5)). Two major explanations are (i) direct electron transfer from b(5) and (ii) a conformational effect in the absence of electron transfer. Some P450s (e.g. 3A4, 2C9, 17A, and 4A7) are stimulated by either b(5) or b(5) devoid of heme (apo-b(5)), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated by b(5) but not by apo-b(5). Recently, a proposal has been made by Guryev et al. (Biochemistry 40, 5018-5031, 2001) that the stimulation by apo-b(5) can be explained only by transfer of heme from P450 preparations to apo-b(5), enabling electron transfer. We have repeated earlier findings of stimulation of catalytic activity of testosterone 6beta-hydroxylation activities with four P450 preparations, in which nearly all of the heme was accounted for as P450. Spectral analysis of mixtures indicated that only approximately 5% of the heme can be transferred to apo-b(5), which cannot account for the observed stimulation. The presence of the heme scavenger apomyoglobin did not inhibit the stimulation of P450 3A4-dependent testosterone or nifedipine oxidation activity. Further evidence against the presence of loosely bound P450 3A4 heme was provided in experiments with apo-heme oxygenase, in which only 3% of the P450 heme was converted to biliverdin. Finally, b(5) supported NADH-b(5) reductase/P450 3A4-dependent testosterone 6beta-hydroxylation, but apo-b(5) did not. Thus, apo-b(5) can stimulate P450 3A4 reactions as well as b(5) in the absence of electron transfer, and heme transfer from P450 3A4 to apo-b(5) cannot be used to explain the catalytic stimulation.
The cooperative binding of Ca2+ ions is an essential functional property of the EF-hand family of Ca2+-binding proteins. To understand how these proteins function, it is essential to characterize intermediate binding states in addition to the apo- and holo-proteins. The three-dimensional solution structure and fast time scale internal motional dynamics of the backbone have been determined for the half-saturated state of the N56A mutant of calbindin D9k with Ca2+ bound only in the N-terminal site. The extent of conformational reorganization and a loss of flexibility in the C-terminal EF-hand upon binding of an ion in the N-terminal EF-hand provide clear evidence of the importance of site-site interactions in this family of proteins, and demonstrates the strength of long-range effects in the cooperative EF-hand Ca2+-binding domain.
The three-dimensional solution structure of apo rabbit lung calcyclin has been refined to high resolution through the use of heteronuclear NMR spectroscopy and 13C, 15N-enriched protein. Upon completing the assignment of virtually all of the 15N, 13C and 1H NMR resonances, the solution structure was determined from a combination of 2814 NOE-derived distance constraints, and 272 torsion angle constraints derived from scalar couplings. A large number of critical inter-subunit NOEs (386) were identified from 13C-select, 13C-filtered NOESY experiments, providing a highly accurate dimer interface. The combination of distance geometry and restrained molecular dynamics calculations yielded structures with excellent agreement with the experimental data and high precision (rmsd from the mean for the backbone atoms in the eight helices: 0.33 A). Calcyclin exhibits a symmetric dimeric fold of two identical 90 amino acid subunits, characteristic of the S100 subfamily of EF-hand Ca(2+)-binding proteins. The structure reveals a readily identified pair of putative sites for binding of Zn2+. In order to accurately determine the structural features that differentiate the various S100 proteins, distance difference matrices and contact maps were calculated for the NMR structural ensembles of apo calcyclin and rat and bovine S100B. These data show that the most significant variations among the structures are in the positioning of helix III and in loops, the regions with least sequence similarity. Inter-helical angles and distance differences for the proteins show that the positioning of helix III of calcyclin is most similar to that of bovine S100B, but that the helix interfaces are more closely packed in calcyclin than in either S100B structure. Surprisingly large differences were found in the positioning of helix III in the two S100B structures, despite there being only four non-identical residues, suggesting that one or both of the S100B structures requires further refinement.
Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-beta1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-beta1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-beta1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-beta1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of beta-actin mRNA. SP transcription rates after 24 h of exposure to TGF-beta1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-beta1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-beta1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-beta1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.