Hydrogen/Deuterium Exchange Mass Spectrometry Provides Insights into the Role of Drosophila Testis-Specific Myosin VI Light Chain AndroCaM.

In Drosophila testis, myosin VI plays a special role, distinct from its motor function, by anchoring components to the unusual actin-based structures (cones) that are required for spermatid individualization. For this, the two calmodulin (CaM) light-chain molecules of myosin VI are replaced by androcam (ACaM), a related protein with 67% identity to CaM. Although ACaM has a similar bi-lobed structure to CaM, with two EF hand-type Ca2+ binding sites per lobe, only one functional Ca2+ binding site operates in the amino-terminus. To understand this light chain substitution, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to examine dynamic changes in ACaM and CaM upon Ca2+ binding and interaction with the two CaM binding motifs of myosin VI (insert2 and IQ motif). HDX-MS reveals that binding of Ca2+ to ACaM destabilizes its N-lobe but stabilizes the entire C-lobe, whereas for CaM, Ca2+ binding induces a pattern of alternating stabilization/destabilization throughout. The conformation of this stable holo-C-lobe of ACaM seems to be a "prefigured" version of the conformation adopted by the holo-C-lobe of CaM for binding to insert2 and the IQ motif of myosin VI. Strikingly, the interaction of holo-ACaM with either peptide converts the holo-N-lobe to its Ca2+-free, more stable, form. Thus, ACaM in vivo should bind the myosin VI light chain sites in an apo-N-lobe/holo-C-lobe state that cannot fulfill the Ca2+-related functions of holo-CaM required for myosin VI motor assembly and activity. These findings indicate that inhibition of myosin VI motor activity is a precondition for transition to an anchoring function.

Nipah Virus V Protein Binding Alters MDA5 Helicase Folding Dynamics.

Nipah virus (NiV) is an emerging and deadly zoonotic paramyxovirus that is responsible for periodic epidemics of acute respiratory illness and encephalitis in humans. Previous studies have shown that the NiV V protein antagonizes host antiviral immunity, but the molecular mechanism is incompletely understood. To address this gap, we biochemically characterized NiV V binding to the host pattern recognition receptor MDA5. We find that the C-terminal domain of NiV V (VCTD) is sufficient to bind the MDA5SF2 domain when recombinantly co-expressed in bacteria. Analysis by hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies revealed that NiV VCTD is conformationally dynamic, and binding to MDA5 reduces the dynamics of VCTD. Our results also suggest that the ß-sheet region in between the MDA5 Hel1, Hel2, and Hel2i domains exhibits rapid HDX. Upon VCTD binding, these ß-sheet and adjacent residues show significant protection. Collectively, our findings suggest that NiV V binding disrupts the helicase fold and dynamics of MDA5 to antagonize host antiviral immunity.

Domain-specific biochemical and serological characterization of SARS-CoV-2 nucleocapsid protein.

Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

Characterization of SARS-CoV-2 nucleocapsid protein reveals multiple functional consequences of the C-terminal domain.

Nucleocapsid (N) encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays key roles in the replication cycle and is a critical serological marker. Here, we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high-affinity RNA-binding platform. We also map the RNA-binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose coils, showing how N-RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based on antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers.

Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain.

Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

Isolation of Amine Derivatives of (ZnSe)34 and (CdTe)34. Spectroscopic Comparisons of the (II-VI)13 and (II-VI)34 Magic-Size Nanoclusters.

The spectroscopically observed magic-size nanoclusters (ZnSe)34 and (CdTe)34 are isolated as amine derivatives. The nanoclusters [(ZnSe)34( n-octylamine)29±6(di- n-octylamine)5±4] and [(CdTe)34( n-octylamine)4±3(di- n-pentylamine)13±3] are fully characterized by combustion-based elemental analysis, UV-visible spectroscopy, IR spectroscopy, and mass spectrometry. Amine derivatives of both (ZnSe)34 and (CdTe)34 are observed to convert to the corresponding (ZnSe)13 and (CdTe)13 derivatives, indicating that the former are kinetic products and the latter thermodynamic products, under the conditions employed. This conversion process is significantly inhibited in the presence of secondary amines. The isolation of the two new nanocluster derivatives adds to a total of nine of 12 possible isolated derivatives in the (II-VI)13 and (II-VI)34 families (II = Zn, Cd; VI = S, Se, Te), allowing comparisons of their properties. The members of these two families exhibit extensive spectroscopic homologies. In both the (II-VI)13 and (II-VI)34 families, linear relationships are established between the lowest-energy nanocluster electronic transition and the band gap of the corresponding bulk semiconductor phase.

The Magic-Size Nanocluster (CdSe)34 as a Low-Temperature Nucleant for Cadmium Selenide Nanocrystals; Room-Temperature Growth of Crystalline Quantum Platelets.

Reaction of Cd(OAc)2·2H2O and selenourea in primary-amine/secondary-amine cosolvent mixtures affords crystalline CdSe quantum platelets at room temperature. Their crystallinity is established by X-ray diffraction analysis (XRD), high-resolution transmission electron microscopy (TEM), and their sharp extinction and photoluminescence spectra. Reaction monitoring establishes the magic-size nanocluster (CdSe)34 to be a key intermediate in the growth process, which converts to CdSe quantum platelets by first-order kinetics with no induction period. The results are interpreted to indicate that the critical crystal-nucleus size for CdSe under these conditions is in the range of (CdSe)34 to (CdSe)68. The nanocluster is obtained in isolated form as [(CdSe)34(n-octylamine)16(di-n-pentylamine)2], which is proposed to function as crystal nuclei that may be stored in a bottle.

The IC138 and IC140 intermediate chains of the I1 axonemal dynein complex bind directly to tubulin.

Dyneins are minus end directed microtubule motors that play a critical role in ciliary and flagellar movement. Ciliary dyneins, also known as axonemal dyneins, are characterized based on their location on the axoneme, either as outer dynein arms or inner dynein arms. The I1 dynein is the best-characterized subspecies of the inner dynein arms; however the interactions between many of the components of the I1 complex and the axoneme are not well defined. In an effort to elucidate the interactions in which the I1 components are involved, we performed zero-length crosslinking on axonemes and studied the crosslinked products formed by the I1 intermediate chains, IC138 and IC140. Our data indicate that IC138 and IC140 bind directly to microtubules. Mass-spectrometry analysis of the crosslinked product identified both α- and ß-tubulin as the IC138 and IC140 binding partners. This was further confirmed by crosslinking experiments carried out on purified I1 fractions bound to Taxol-stabilized microtubules. Furthermore, the interaction between IC140 and tubulin is lost when IC138 is absent. Our studies support previous findings that intermediate chains play critical roles in the assembly, axonemal targeting and regulation of the I1 dynein complex.

Preparation of primary amine derivatives of the magic-size nanocluster (CdSe)13.

Four [(CdSe)13(RNH2)13] derivatives (R = n-propyl, n-pentyl, n-octyl, and oleyl) are prepared by reaction of Cd(OAc)2·2H2O and selenourea in the corresponding primary-amine solvent. Nanoclusters grow in spontaneously formed amine-bilayer templates and are characterized by elemental analysis, IR spectroscopy, UV-vis spectroscopy, TEM, and low-angle XRD. Derivative [(CdSe)13(n-propylamine)13] is isolated as a yellowish-white solid (MP 98 °C) on the gram scale. These compounds are the first derivatives of magic-size CdSe nanoclusters to be isolated in purity.

Identification of potential mediators of retinotopic mapping: a comparative proteomic analysis of optic nerve from WT and Phr1 retinal knockout mice.

Retinal ganglion cells (RGCs) transmit visual information topographically from the eye to the brain, creating a map of visual space in retino-recipient nuclei (retinotopy). This process is affected by retinal activity and by activity-independent molecular cues. Phr1, which encodes a presumed E3 ubiquitin ligase (PHR1), is required presynaptically for proper placement of RGC axons in the lateral geniculate nucleus and the superior colliculus, suggesting that increased levels of PHR1 target proteins may be instructive for retinotopic mapping of retinofugal projections. To identify potential target proteins, we conducted a proteomic analysis of optic nerve to identify differentially abundant proteins in the presence or absence of Phr1 in RGCs. 1D gel electrophoresis identified a specific band in controls that was absent in mutants. Targeted proteomic analysis of this band demonstrated the presence of PHR1. Additionally, we conducted an unbiased proteomic analysis that identified 30 proteins as being significantly different between the two genotypes. One of these, heterogeneous nuclear ribonucleoprotein M (hnRNP-M), regulates antero-posterior patterning in invertebrates and can function as a cell surface adhesion receptor in vertebrates. Thus, we have demonstrated that network analysis of quantitative proteomic data is a useful approach for hypothesis generation and for identifying biologically relevant targets in genetically altered biological models.

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium.

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/µg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.

Top-down mass spectrometry: recent developments, applications and perspectives.

Top-down mass spectrometry is an emerging approach for the analysis of intact proteins. The term was coined as a contrast with the better-established, bottom-up strategy for analysis of peptide fragments derived from digestion, either enzymatically or chemically, of intact proteins. Although the term top-down originates from proteomics, it can also be applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein assemblies or complexes. Traditionally, mass spectrometry has usually started with intact molecules, and in this regard, top-down approaches reflect the spirit of mass spectrometry. This article provides an overview of the methodologies in top-down mass spectrometry and then reviews applications covering protein posttranslational modifications, protein biophysics, DNAs/RNAs, and protein assemblies. Finally, challenges and future directions are discussed.

Identification of CaMKII phosphorylation sites in Connexin43 by high-resolution mass spectrometry.

Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function, and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multifunctional protein kinase, Ca(2+)/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca(2+) homeostasis, transcription, apoptosis, and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO(2), and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these "orthogonal" methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.

Improved mass spectrometric characterization of protein glycosylation reveals unusual glycosylation of maize-derived bovine trypsin.

Although bottom-up proteomics using tryptic digests is widely used to locate post-translational modifications (PTM) in proteins, there are cases where the protein has several potential modification sites within a tryptic fragment and MS(2) strategies fail to pinpoint the location. We report here a method using two proteolytic enzymes, trypsin and pepsin, in combination followed by tandem mass spectrometric analysis to provide fragments that allow one to locate the modification sites. We used this strategy to find a glycosylation site on bovine trypsin expressed in maize (TrypZean). Several glycans are present, and all are attached to a nonconsensus N-glycosylation site on the protein.

Layered Calcium Structures of p-Phosphonic Acid O-Methyl-Calix[6]arene.

Hexamethoxy-calix[6]arene has been fully functionalized with p-phosphonic acid groups on the upper rim in 57% yield over three steps, and has been authenticated in the solid state by X-ray diffraction as either a nitrate salt or one of two calcium complexes. The latter differ by the ratio of calcium ions per calixarene, either 3:1 or 4:1. In both structures the coordination sphere of the calcium ions is made up of oxygen atoms from the phosphonic acid groups and from water of crystallization, as part of extended polymeric layers in the extended 3D packing. Hirshfeld surface analysis shows extensive O...H and O...Ca interactions for the phosphonic acid moieties in both calcium structures. MALDI-TOF MS of the hexaphosphonic acid shows nano-arrays consisting of up to a maximum of 28 calixarene units.

A single peptide-MHC complex positively selects a diverse and specific CD8 T cell repertoire.

Pathogen recognition by T cells is dependent on their exquisite specificity for self-major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide-MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.

Proteomic analyses of native brain K(V)4.2 channel complexes.

Somatodendritic A-type (I(A)) voltage-gated K(+) (K(V)) channels are key regulators of neuronal excitability, functioning to control action potential waveforms, repetitive firing and the responses to synaptic inputs. Rapidly activating and inactivating somatodendritic I(A) channels are encoded by K(V)4 alpha subunits and accumulating evidence suggests that these channels function as components of macromolecular protein complexes. Mass spectrometry (MS)-based proteomic approaches were developed and exploited here to identify potential components and regulators of native brain K(V)4.2-encoded I(A) channel complexes. Using anti-K(V)4.2 specific antibodies, K(V)4.2 channel complexes were immunoprecipitated from adult wild type mouse brain. Parallel control experiments were performed on brain samples isolated from (K(V)4.2(-/-)) mice harboring a targeted disruption of the KCND2 (K(V)4.2) locus. Three proteomic strategies were employed: an in-gel approach, coupled to one-dimensional liquid chromatography-tandem MS (1D-LC-MS/MS), and two in-solution approaches, followed by 1D- or 2D-LC-MS/MS. The targeted in-gel 1D-LC-MS/MS analyses demonstrated the presence of the K(V)4 alpha subunits (K(V)4.2, K(V)4.3 and K(V)4.1) and the K(V)4 accessory, KChIP (KChIP1-4) and DPP (DPP6 and 10), proteins in native brain K(V)4.2 channel complexes. The more comprehensive, in-solution approach, coupled to 2D-LC-MS/MS, also called Multidimensional Protein Identification Technology (MudPIT), revealed that additional regulatory proteins, including the K(V) channel accessory subunit K(V)beta1, are also components of native brain K(V)4.2 channel complexes. Additional biochemical and functional approaches will be required to elucidate the physiological roles of these newly identified K(V)4 interacting proteins.

Combined charged residue-field emission model of macromolecular electrospray ionization.

The mechanism of the multiple charging of macromolecules in electrospray ionization (ESI) continues to inspire debate and controversy. Recently, we proposed that the number of charges on a macromolecule is determined by the emission of small charge carriers from macromolecule-containing nanodroplets and that, after solvent evaporation, the remaining charge is transferred to the macromolecule. In this study, we tested the applicability of this new theory for macromolecular, positive-ion ESI mass spectrometry by measuring the mean charge states and charge distributions of globular proteins under non-denaturing and denaturing conditions. Predictions of protein mean charge states for native state proteins are in excellent agreement with mass spectrometric measurements, giving strong credence to the proposed theory. Theoretical predictions are also in good agreement with mean charge states measured for proteins in basic solutions (pH = 11.5). For some proteins in acidic solutions (pH = 2.1), the measured mean charge states are anomalously higher than the Rayleigh limit of a water droplet with a volume equivalent to that of the protein. We propose that some macromolecules that are highly charged in solution may desorb from charged droplets of the same polarity in a similar manner to that whereby charge carriers emit from nanodroplets, leading to "supercharged" macromolecular ions. Examination of rate expressions for solvent evaporation and charge-carrier emission demonstrates that the newly proposed model for ESI is consistent with previously reported ion-emission kinetics. Overall, we obtained additional experimental evidence for the charge carrier emission model for macromolecular charging, suggesting opportunities for understanding and applying ESI-MS.

Radical Cation/Radical Reactions: A Fourier Transform Ion Cyclotron Resonance Study of Allyl Radical Reacting with Aromatic Radical Cations.

A method for the study of reactions of open-shell neutrals (radicals) and radical cations is described. Pyrolysis (25-1500 degrees C) of thermally labile compounds, such as, 1,5-hexadiene via a Chen nozzle yields a seeded beam of reactive species in helium. The pyrolysis products are then analyzed by electron ionization (EI) or reacted with stored ions. Electron ionization of the pyrolysis products of 1,5-hexadiene shows that both the allyl radical and allene are generated. Reactions of benzene radical cations and the pyrolysis products of 1,5-hexadiene result in carbon-carbon bond formation. Those reactions of allyl radical with the benzene radical cation yield the C(7)H(7) (+) ion of m/z 91, permitting an unusual entry into arenium ions. The reaction of allene with benzene radical cation in contrast yields C(9)H(10) (+). and C(9)H(9) (+).