585 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
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
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 Ă… 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
The dynamic nature of gene expression enables cellular programming, homeostasis, and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high precision spatiotemporal control of many cellular functions1-11. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here, we describe the development of Light-Inducible Transcriptional Effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain12-14 with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical co-factors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility3,4,6,7,15. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of awake mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states
Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions
Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation
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