711 research outputs found
Sulfolane-Induced Supercharging of Electrosprayed Salt Clusters: An Experimental/Computational Perspective.
It is well-known that supercharging agents (SCAs) such as sulfolane enhance the electrospray ionization (ESI) charge states of proteins, although the mechanistic origins of this effect remain contentious. Only very few studies have explored SCA effects on analytes other than proteins or peptides. This work examines how sulfolane affects electrosprayed NaI salt clusters. Such alkali metal halide clusters have played a key role for earlier ESI mechanistic studies, making them interesting targets for supercharging investigations. ESI of aqueous NaI solutions predominantly generated singly charged [NanI(n-1)]+ clusters. The addition of sulfolane resulted in abundant doubly charged [NanI(n-2)Sulfolanes]2+ species. These experimental data for the first time demonstrate that electrosprayed salt clusters can undergo supercharging. Molecular dynamics (MD) simulations of aqueous ESI nanodroplets containing Na+/I- with and without sulfolane were conducted to obtain atomistic insights into the supercharging mechanism. The simulations produced [NanIi]z+ and [NanIiSulfolanes]z+ clusters similar to those observed experimentally. The MD trajectories demonstrated that these clusters were released into the gas phase upon droplet evaporation to dryness, in line with the charged residue model. Sulfolane was found to evaporate much more slowly than water. This slow evaporation, in conjunction with the large dipole moment of sulfolane, resulted in electrostatic stabilization of the shrinking ESI droplets and the final clusters. Hence, charge-dipole stabilization causes the sulfolane-containing droplets and clusters to retain more charge, thereby providing the mechanistic foundation of salt cluster supercharging
Enhancing Protein Electrospray Charge States by Multivalent Metal Ions: Mechanistic Insights from MD Simulations and Mass Spectrometry Experiments.
The structure and reactivity of electrosprayed protein ions is governed by their net charge. Native proteins in non-denaturing aqueous solutions produce low charge states. More highly charged ions are formed when electrospraying proteins that are unfolded and/or exposed to organic supercharging agents. Numerous studies have explored the electrospray process under these various conditions. One phenomenon that has received surprisingly little attention is the charge enhancement caused by multivalent metal ions such as La3+ when electrospraying proteins out of non-denaturing solutions. Here, we conducted mass spectrometry and ion mobility spectrometry experiments, in combination with molecular dynamics (MD) simulations, to uncover the mechanistic basis of this charge enhancement. MD simulations of aqueous ESI droplets reproduced the experimental observation that La3+ boosts protein charge states relative to monovalent metals (e.g., Na+). The simulations showed that gaseous proteins were released by solvent evaporation to dryness, consistent with the charged residue model. Metal ion ejection kept the shrinking droplets close to the Rayleigh limit until ∼99% of the solvent had left. For droplets charged with Na+, metal adduction during the final stage of solvent evaporation produced low protein charge states. Droplets containing La3+ showed a very different behavior. The trivalent nature of La3+ favored adduction to the protein at a very early stage, when most of the solvent had not evaporated yet. This irreversible binding via multidentate contacts suppressed La3+ ejection from the vanishing droplets, such that the resulting gaseous proteins carried significantly more charge. Our results illustrate that MD simulations are suitable for uncovering intricate aspects of electrospray mechanisms, paving the way toward an atomistic understanding of mass spectrometry based analytical workflows
Gas Phase Protein Folding Triggered by Proton Stripping Generates Inside-Out Structures: A Molecular Dynamics Simulation Study.
The properties of electrosprayed protein ions continue to be enigmatic, owing to the absence of high-resolution structure determination methods in the gas phase. There is considerable evidence that under properly optimized conditions these ions preserve solution-like conformations and interactions. However, it is unlikely that these solution-like conformers represent the intrinsic structural preferences of gaseous proteins. In an effort to uncover what such intrinsically preferred conformers might look like, we performed molecular dynamics (MD) simulations of gaseous ubiquitin. Our work was inspired by recent gas phase experiments, where highly extended 13+ ubiquitin ions were transformed to compact 3+ species by proton stripping (Laszlo, K. J.; Munger, E. B.; Bush, M. F. J. Am. Chem. Soc. 2016, 138, 9581-9588). Our simulations covered several microseconds and used a mobile-proton algorithm to account for the fact that a H+ in gaseous proteins can migrate between different titratable sites. Proton stripping caused folding of ubiquitin into heterogeneous inside-out structures. The hydrophilic core of these conformers was stabilized by charge-charge and polar interactions, while hydrophobic residues were located on the protein surface. Collision cross sections of these MD structures were in good agreement with experimental results. The inside-out structures generated during gas phase folding are in striking contrast to the solution behavior which is dominated by the hydrophobic effect, i.e., the tendency to bury hydrophobic side chains in the core (instead of exposing them to the surface). We do not dispute that native-like proteins can be transferred into the gas phase as kinetically trapped species. However, those metastable conformers do not represent the intrinsic structural preferences of gaseous proteins. Our work for the first time provides detailed insights into the properties of intrinsically preferred gas phase conformers, and we unequivocally find them to have inside-out architectures
Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome
Many aspects of protein function rely on conformational fluctuations. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) provides a window into these dynamics. Despite the widespread use of HDX-MS, it remains unclear whether this technique provides a truly comprehensive view of protein dynamics. HDX is mediated by H-bond-opening/closing events, implying that HDX methods provide an H-bond-centric view. This raises the question if there could be fluctuations that leave the H-bond network unaffected, thereby rendering them undetectable by HDX-MS. We explore this issue in experiments on cytochrom
Interrogating the Quaternary Structure of Noncanonical Hemoglobin Complexes by Electrospray Mass Spectrometry and Collision-Induced Dissociation.
Various activation methods are available for the fragmentation of gaseous protein complexes produced by electrospray ionization (ESI). Such experiments can potentially yield insights into quaternary structure. Collision-induced dissociation (CID) is the most widely used fragmentation technique. Unfortunately, CID of protein complexes is dominated by the ejection of highly charged monomers, a process that does not yield any structural insights. Using hemoglobin (Hb) as a model system, this work examines under what conditions CID generates structurally informative subcomplexes. Native ESI mainly produced tetrameric Hb ions. In addition, noncanonical hexameric and octameric complexes were observed. CID of all these species [(αβ)2, (αβ)3, and (αβ)4] predominantly generated highly charged monomers. In addition, we observed hexamer → tetramer + dimer dissociation, implying that hexamers have a tetramer··dimer architecture. Similarly, the observation of octamer → two tetramer dissociation revealed that octamers have a tetramer··tetramer composition. Gas-phase candidate structures of Hb assemblies were produced by molecular dynamics (MD) simulations. Ion mobility spectrometry was used to identify the most likely candidates. Our data reveal that the capability of CID to produce structurally informative subcomplexes depends on the fate of protein-protein interfaces after transfer into the gas phase. Collapse of low affinity interfaces conjoins the corresponding subunits and favors CID via monomer ejection. Structurally informative subcomplexes are formed only if low affinity interfaces do not undergo a major collapse. However, even in these favorable cases CID is still dominated by monomer ejection, requiring careful analysis of the experimental data for the identification of structurally informative subcomplexes
Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation
The E3 ligase parkin ubiquitinates outer mitochondrial membrane
proteins during oxidative stress and is linked to early-onset
Parkinson’s disease. Parkin is autoinhibited but is activated by the
kinase PINK1 that phosphorylates ubiquitin leading to parkin
recruitment, and stimulates phosphorylation of parkin’s N-terminal
ubiquitin-like (pUbl) domain. How these events alter the
structure of parkin to allow recruitment of an E2~Ub conjugate
and enhanced ubiquitination is an unresolved question. We
present a model of an E2~Ub conjugate bound to the phosphoubiquitin-loaded
C-terminus of parkin, derived from NMR chemical
shift perturbation experiments. We show the UbcH7~Ub conjugate
binds in the open state whereby conjugated ubiquitin binds to the
RING1/IBR interface. Further, NMR and mass spectrometry experiments
indicate the RING0/RING2 interface is re-modelled,
remote from the E2 binding site, and this alters the reactivity of
the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer.
Our experiments provide evidence that parkin phosphorylation
and E2~Ub recruitment act synergistically to enhance a weak
interaction of the pUbl domain with the RING0 domain and rearrange
the location of the RING2(Rcat) domain to drive parkin
activity
Kennis delen onder leraren: Een onderzoek naar de relaties tussen Occupational Self-Efficacy, Werk bevlogenheid, Human Resource Management en Kennis delen
Knowledge sharing is one of the professionalizetion processes and is an important factor in the competition between organizations and for innovation processes to sustain. In this study the central theme is the way knowledge sharing is affected by occupational self-efficacy (OSE), work engagement and High Commitment HRM (HC-HRM). In investigating these relations the AMO framework is used. The research data were obtained by 126 teachers from one secondary school. However from the regression analyses it was learned that the relationship between the variables OSE, HC HRM and work engagement with knowledge sharing was more complex than expected. Additional analyses by means of a three-way interaction analysis suggests that the combination of high experienced HC-HRM and low experienced OSE or the other way around is, related to more knowledge sharing. The findings are important for managers who want to promote processes of knowledge sharing in their school organization
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA
The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Ã… resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.National Institutes of Health (U.S.) (Grant 5DP1-MH100706
Optical control of mammalian endogenous transcription and epigenetic states
A theoretical underpinning of the standard model of fundamental particles and interactions is CPT invariance, which requires that the laws of physics be invariant under the combined discrete operations of charge conjugation, parity and time reversal. Antimatter, the existence of which was predicted by Dirac, can be used to test the CPT theorem—experimental investigations involving comparisons of particles with antiparticles are numerous. Cold atoms and anti-atoms, such as hydrogen and antihydrogen, could form the basis of a new precise test, as CPT invariance implies that they must have the same spectrum. Observations of antihydrogen in small quantities and at high energies have been reported at the European Organization for Nuclear Research (CERN) and at Fermilab, but these experiments were not suited to precision comparison measurements. Here we demonstrate the production of antihydrogen atoms at very low energy by mixing trapped antiprotons and positrons in a cryogenic environment. The neutral anti-atoms have been detected directly when they escape the trap and annihilate, producing a characteristic signature in an imaging particle detector
Computed tomography-osteoabsorptiometry for assessing the density distribution of subchondral bone as a measure of long-term mechanical adaptation in individual joints
To estimate subchondral mineralisation patterns which represent the long-term loading history of individual joints, a method has been developed employing computed tomography (CT) which permits repeated examination of living joints. The method was tested on 5 knee, 3 sacroiliac, 3 ankle and 5 shoulder joints and then investigated with X-ray densitometry. A CT absorptiometric presentation and maps of the area distribution of the subchondral bone density areas were derived using an image analyser. Comparison of the results from both X-ray densitometry and CT-absorptiometry revealed almost identical pictures of distribution of the subchondral bone density. The method may be used to examine subchondral mineralisation as a measure of the mechanical adaptability of joints in the living subject
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