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Most downloaded bioRxiv papers, all time

in category biochemistry

2,039 results found. For more information, click each entry to expand.

81: A Multi-Crystal Method for Extracting Obscured Signal from Crystallographic Electron Density
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Posted to bioRxiv 05 Sep 2016

A Multi-Crystal Method for Extracting Obscured Signal from Crystallographic Electron Density
1,496 downloads biochemistry

Nicholas M Pearce, Anthony R. Bradley, Patrick Collins, Tobias Krojer, Radoslaw P Nowak, Romain Talon, Brian D Marsden, Sebastian Kelm, Jiye Shi, Charlotte M. Deane, Frank von Delft

Macromolecular crystallography is relied on to reveal subtle atomic difference between samples (e.g. ligand binding); yet their detection and modelling is subjective and ambiguous density is experimentally common, since molecular states of interest are generally only fractionally present. The existing approach relies on careful modelling for maximally accurate maps to make contributions of the minor fractions visible; in practice, this is time-consuming and non-objective. Instead, our PanDDA method automatically reveals clear electron density for only the changed state, even from poor models and inaccurate maps, by subtracting a proportion of the confounding ground state, accurately estimated by averaging many ground state crystals. Changed states are objectively identifiable from statistical distributions of density values; arbitrarily large searches are thus automatable. The method is completely general, implying new best practice for all changed-state studies. Finally, we demonstrate the incompleteness of current atomic models, and the need for new multi-crystal deconvolution paradigms.

82: A well-controlled BioID design for endogenous bait proteins
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Posted to bioRxiv 26 Sep 2018

A well-controlled BioID design for endogenous bait proteins
1,488 downloads biochemistry

Giel Vandemoortele, Delphine De Sutter, Aline Moliere, Jarne Pauwels, Kris Gevaert, Sven Eyckerman

The CRISPR/Cas9 revolution is profoundly changing the way life sciences technologies are used. Many assays now rely on engineered clonal cell lines to eliminate overexpression of bait proteins. Control cell lines are typically non-engineered cells or engineered clones implying a considerable risk for artefacts because of clonal variation. Genome engineering can also transform BioID, a proximity labelling method that relies on fusing a bait protein to a promiscuous biotin ligase, BirA*, resulting in the tagging of vicinal proteins. We here propose an innovative design to enable BioID for endogenous proteins wherein we introduce a T2A-BirA* module at the C-terminus of endogenous p53 by genome engineering, leading to bi-cistronic expression of both p53 and BirA* under control of the endogenous promoter. By targeting a Cas9-cytidine deaminase base editor to the T2A auto-cleavage site, we can efficiently derive an isogenic population expressing a functional p53-BirA* fusion protein. Using quantitative proteomics we show significant benefits over classical ectopic expression of p53-BirA*, and we provide a first well-controlled view on the proximal proteins of endogenous p53 in colon carcinoma cells. This novel application for base editors expands the CRISPR/Cas9 toolbox and can be a valuable addition for synthetic biology.

83: Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution
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Posted to bioRxiv 28 May 2018

Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution
1,481 downloads biochemistry

Christian D. Kelstrup, Konstantin Aizikov, Tanveer S. Batth, Arne Kreutzman, Dmitry Grinfeld, Oliver Lange, Daniel Mourad, Alexander Makarov, Jesper V. Olsen

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer masses.

84: ATOMIC STRUCTURES OF GLUCOSE, FRUCTOSE AND SUCROSE AND EXPLANATION OF ANOMERIC CARBON
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Posted to bioRxiv 23 Jan 2014

ATOMIC STRUCTURES OF GLUCOSE, FRUCTOSE AND SUCROSE AND EXPLANATION OF ANOMERIC CARBON
1,479 downloads biochemistry

Raji Heyrovska

Presented here are the structures of three biologically important sweet sugars, based on the additivity of covalent atomic radii in bond lengths. The observed smaller carbon-oxygen distances involving the 'anomeric' carbons of the open chain hexoses are explained here, for the first time, as due to the smaller covalent double bond radii of carbon and oxygen than their single bond radii in the cyclic forms and in sucrose. The atomic structures of all the three carbohydrates, drawn to scale in colour, have been presented here also for the first time.

85: Exploring DCAF15 for reprogrammable targeted protein degradation
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Posted to bioRxiv 08 Feb 2019

Exploring DCAF15 for reprogrammable targeted protein degradation
1,453 downloads biochemistry

Seemon Coomar, Dennis G. Gillingham

The targeted degradation of proteins by reprogramming E3 ligases with bifunctional small molecules is an exciting area of chemical biology because it promises a set of chemical tools for achieving the same task as siRNA or CRISPR/Cas9. Although there are hundreds of E3 ligases in the human proteome only a few have been shown to be reprogrammable to target new proteins. Recently various arylsulfonamides were shown to induce degradation of the splicing factor RBM39 via the RING type E3 ligase CRL4DCAF15. Here we identify the arylsulfonamide most amenable to chemical modifications and demonstrate its behaviour in bifunctional reprogramming.

86: Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15
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Posted to bioRxiv 16 Aug 2019

Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15
1,447 downloads biochemistry

Tyler Faust, Hojong Yoon, Radosław P. Nowak, Katherine A Donovan, Zhengnian Li, Quan Cai, Nicholas A Eleuteri, Tinghu Zhang, Nathanael S. Gray, Eric S. Fischer

The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the Cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at 4.4 angstrom resolution, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a novel fold stabilized by DDA1, and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrates how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders.

87: PhoX - an IMAC-enrichable Crosslinking Reagent
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Posted to bioRxiv 21 Feb 2019

PhoX - an IMAC-enrichable Crosslinking Reagent
1,447 downloads biochemistry

Barbara A. Steigenberger, Roland J. Pieters, Albert J. R. Heck, Richard A. Scheltema

Chemical crosslinking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, sub-stoichiometric reaction efficiencies render routine detection of crosslinked peptides problematic. Here we present a new tri-functional crosslinking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the crosslinked peptides amenable to the well-established IMAC enrichment. The handle allows for 300x enrichment efficiency and 97% specificity, dramatically reducing measurement time and improving data quality. We exemplify the approach on various model proteins and protein complexes, e.g. resulting in a structural model of the LRP1/RAP complex. PhoX is also applicable to whole cell lysates. When focusing the database search on ribosomal proteins, our first attempt resulted in 355 crosslinks, out-performing current efforts in less measurement time.

88: m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins
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Posted to bioRxiv 07 Jul 2019

m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins
1,430 downloads biochemistry

Ye Fu, Xiaowei Zhuang

Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of m6A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m6A-binding YTHDF proteins in regulating phase separation.

89: Programmable RNA recognition using a CRISPR-associated Argonaute
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Posted to bioRxiv 23 Oct 2017

Programmable RNA recognition using a CRISPR-associated Argonaute
1,425 downloads biochemistry

Audrone Lapinaite, Jennifer A. Doudna, Jamie H. D. Cate

Argonaute proteins (Agos) are present in all domains of life. While the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci and use non-canonical 5'-hydroxyled guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5' nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5'-BrdU modified gRNAs, we mapped the seed region of the gRNA, and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the 6th and 7th positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific Adenosine to Inosine edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5'-BrdU modified gRNAs can be used as a highly-specific RNA-targeting platform to probe RNA biology.

90: Targeted DNA transposition using a dCas9-transposase fusion protein
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Posted to bioRxiv 08 Mar 2019

Targeted DNA transposition using a dCas9-transposase fusion protein
1,419 downloads biochemistry

Shivam Bhatt, Ronald Chalmers

Homology directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Catalytically inactive Cas9 is attractive candidate for targeting a transposase fusion-protein because of its high specificity and affinity for its binding site. Here we demonstrate efficient Cas9 targeting of a mariner transposon. Targeted integrations were tightly constrained at two adjacent TA dinucleotides about 20 bp to one side of the gRNA binding site. Biochemical analysis of the nucleoprotein complexes demonstrated that the transposase and Cas9 moieties of the fusion protein can bind their respective substrates independently. In the presence of the Cas9 target DNA, kinetic analysis revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase behaved normally and was proficient for integration in vitro and in vivo.

91: Riboglow: a multicolor riboswitch-based platform for live cell imaging of mRNA and small non-coding RNA in mammalian cells
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Posted to bioRxiv 10 Oct 2017

Riboglow: a multicolor riboswitch-based platform for live cell imaging of mRNA and small non-coding RNA in mammalian cells
1,410 downloads biochemistry

Esther Braselmann, Aleksandra J Wierzba, Jacob T Polaski, Mikołaj Chromiński, Zachariah E. Holmes, Sheng-Ting Hung, Dilara Batan, Joshua R. Wheeler, Roy Parker, Ralph Jimenez, Dorota Gryko, Robert T. Batey, Amy E. Palmer

RNAs directly regulate a vast array of critical cellular processes, emphasizing the need for robust approaches to fluorescently tag and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular "Riboglow" platform leverages different color fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescent turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small non-coding U RNA in live mammalian cells. A direct side-by-side comparison revealed that Riboglow outperformed the dye binding aptamer Broccoli and performed on par with the current gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

92: On the validity and errors of the pseudo-first-order kinetics in ligand-receptor binding
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Posted to bioRxiv 30 Apr 2016

On the validity and errors of the pseudo-first-order kinetics in ligand-receptor binding
1,405 downloads biochemistry

Wylie Stroberg, Santiago Schnell

The simple bimolecular ligand-receptor binding interaction is often linearized by assuming pseudo-first-order kinetics when one species is present in excess. Here, a phase-plane analysis allows the derivation of a new condition for the validity of pseudo-first-order kinetics that is independent of the initial receptor concentration. The validity of the derived condition is analyzed from two viewpoints. In the first, time courses of the exact and approximate solutions to the ligand--receptor rate equations are compared when all rate constants are known. The second viewpoint assess the validity through the error induced when the approximate equation is used to estimate kinetic constants from data. Although these two interpretations of validity are often assumed to coincide, we show that they are distinct, and that large errors are possible in estimated kinetic constants, even when the linearized and exact rate equations provide nearly identical solutions.

93: MOHCA-seq: RNA 3D models from single multiplexed proximity-mapping experiments
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Posted to bioRxiv 25 Apr 2014

MOHCA-seq: RNA 3D models from single multiplexed proximity-mapping experiments
1,403 downloads biochemistry

Clarence Yu Cheng, Fang-Chieh Chou, Wipapat Kladwang, Siqi Tian, Pablo Cordero, Rhiju Das

Large RNAs control myriad biological processes but challenge tertiary structure determination. We report that integrating Multiplexed •OH Cleavage Analysis with tabletop deep sequencing (MOHCA-seq) gives nucleotide-resolution proximity maps of RNA structure from single straightforward experiments. After achieving 1-nm resolution models for RNAs of known structure, MOHCA-seq reveals previously unattainable 3D information for ligand-induced conformational changes in a double glycine riboswitch and the sixth community-wide RNA puzzle, an adenosylcobalamin riboswitch.

94: An improved method for the expression screening of membrane protein-GFP fusions in yeast
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Posted to bioRxiv 03 Aug 2017

An improved method for the expression screening of membrane protein-GFP fusions in yeast
1,403 downloads biochemistry

Darren Baldock, Judith Sheldon, Ravi Tailor, Katherine Green, John Ray, Shradha Singh, Kathryn Brocklehurst

The expression and purification of membrane proteins is an extremely challenging area of work within Protein Science. Membrane proteins are required for compound screening and structure determination in industry. Here we describe some new and innovative methodology in developing the membrane protein GFP fusion primary expression screening in yeast. This methodology enables the expression of membrane proteins fused to GFP in both Saccharomyces cerevisiae and Pichia pastoris systems. This capability helps facilitate screening of constructs to establish which are suitable for membrane protein production for compound screening and structure determination. In terms of the primary screening work, we have developed both agar plate and liquid plate expression methodology in yeast. The two approaches correspond well, but the agar plate method is more rapid and we have shown it to have the advantage of allowing cells to be taken directly into confocal microscopy for immediate cell localisation data. Innovative work to extend the methanol induction time in the Pichia agar plate method established good differentiation from the background. A novel agar plate method was also developed for S.cerevisiae which is also presented. These screening methods allow triaging of constructs for either membrane protein preps for biochemical assays or progression to fluorescence size exclusion chromatography; where various detergents can be screened to determine the most appropriate for membrane protein solubilisation, the starting point for purification, crystallisation and structure determination. Membrane targets depicted to demonstrate the improved primary screening methodology are a copper transporter Ctr1p from S. cerevisiae and a water transporter Aqp4 from human origin.

95: Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids.
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Posted to bioRxiv 15 Dec 2016

Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids.
1,402 downloads biochemistry

Joost Snijder, Andrew J Borst, Annie Dosey, Alexandra C Walls, Anika Burrell, Vijay S Reddy, Justin M. Kollman, David Veesler

Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.

96: The meaning of the Michaelis-Menten constant: Km describes a steady-state.
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Posted to bioRxiv 13 Apr 2019

The meaning of the Michaelis-Menten constant: Km describes a steady-state.
1,387 downloads biochemistry

Enric I. Canela, Gemma Navarro, José Luis Beltrán, Rafael Franco

Often, in vitro or in vivo enzyme-mediated catalytic events occur far from equilibrium and, therefore, substrate affinity measured as the inverse of ESDE+S dissociation equilibrium constant (Kd) has a doubtful physiological meaning; in practice it is almost impossible to determine Kd (except using stopped-flow or other sophisticated methodologies). The Michaelis-Menten constant (Km), the concentration of substrate ([S]) providing half of enzyme maximal activity, is not the (Kd). In the simple E+SDES → E+P or in more complex models describing S conversion into P, Km must be considered the constant defining the steady state at any substrate concentration. Enzyme kinetics is based on initial rate determination, i.e. in the linear part of the S to P conversion when the concentration of [ES] remains constant while steady state occurs. We also show that Systems Biology issues such as the time required to respond to a system perturbation, is more dependent on k1, the kinetic constant defining substrate- enzyme association, than on Km. Whereas Km is instrumental for biochemical basic and applied approaches, in any physiological condition, an important parameter to be considered is the substrate association rate (k1).

97: Temperature-responsive competitive inhibition of CRISPR-Cas9
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Posted to bioRxiv 03 Sep 2018

Temperature-responsive competitive inhibition of CRISPR-Cas9
1,380 downloads biochemistry

Fuguo Jiang, Jun-Jie Liu, Beatriz A. Osuna, Michael Xu, Joel D. Berry, Benjamin J. Rauch, Eva Nogales, Joseph Bondy-Denomy, Jennifer A. Doudna

CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory anti-CRISPR (Acr)proteins, including six inhibitors (AcrIIA1-6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its homologue, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially and conditionally.

98: Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex
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Posted to bioRxiv 24 Aug 2017

Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex
1,376 downloads biochemistry

Saikat Chowdhury, Chinatsu Otomo, Alexander Leitner, Kazuto Ohashi, Ruedi Aebersold, Gabriel C. Lander, Takanori Otomo

Autophagy is an enigmatic cellular process in which double-membrane compartments, called autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the phosphatidylinositol-3-phosphate (PI3P)-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore. NOTE: Gabriel C. Lander and Takanori Otomo are both co-corresponding authors.

99: Mechanism-based sirtuin enzyme activation
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Posted to bioRxiv 28 Sep 2015

Mechanism-based sirtuin enzyme activation
1,374 downloads biochemistry

Xiangying Guan, Alok Upadhyay, Sudipto Munshi, Raj Chakrabarti

Sirtuin enzymes are NAD+-dependent protein deacylases that play a central role in the regulation of healthspan and lifespan in organisms ranging from yeast to mammals. There is intense interest in the activation of the seven mammalian sirtuins (SIRT1-7) in order to extend mammalian healthspan and lifespan. However, there is currently no understanding of how to design sirtuin-activating compounds beyond allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates. Moreover, across all families of enzymes, only a dozen or so distinct classes of non-natural small molecule activators have been characterized, with only four known modes of activation among them. None of these modes of activation are based on the unique catalytic reaction mechanisms of the target enzymes. Here, we report a general mode of sirtuin activation that is distinct from the known modes of enzyme activation. Based on the conserved mechanism of sirtuin-catalyzed deacylation reactions, we establish biophysical properties of small molecule modulators that can in principle result in enzyme activation for diverse sirtuins and substrates. Building upon this framework, we propose strategies for the identification, characterization and evolution of hits for mechanism-based enzyme activating compounds. We characterize several small molecules reported in the literature to activate sirtuins besides SIRT1, using a variety of biochemical and biophysical techniques including label-free and labeled kinetic and thermodynamic assays with multiple substrates and protocols for the identification of false positives. We provide evidence indicating that several of these small molecules reported in the published literature are false positives, and identify others as hit compounds for the design of compounds that can activate sirtuins through the proposed mechanism-based mode of action.

100: Native Mass Spectrometry Reveals the Conformational Diversity of the UVR8 Photoreceptor
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Posted to bioRxiv 18 Jul 2018

Native Mass Spectrometry Reveals the Conformational Diversity of the UVR8 Photoreceptor
1,362 downloads biochemistry

Inês S Camacho, Alina Theisen, Linus O Johannissen, L. Aranzazú Díaz-Ramos, John M Christie, Gareth I Jenkins, Bruno Bellina, Perdita Barran, Alex R. Jones

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light resulting in dissociation into monomers, which are considered to be the active state and comprise a β-propeller core domain and intrinsically disordered N- and C-terminal tails. The C-terminus is required for functional binding to signalling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photo-activation. We show that, whilst truncated UVR8 photoconverts from a single conformation of dimers to a single monomer conformation, the full-length protein exist in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C-terminus is primed for activation. In the monomer the extended C-terminus destabilises the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.

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