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Results for “Biochemistry”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 127 records · Page 7Linked to original sources

bioRxiv2015

Structural modeling of the flagellum MS ring protein FliF reveals similarities to the type III secretion system and sporulation complex

The flagellum is a large proteinaceous organelle found at the surface of many bacteria, whose primary role is to allow motility through the rotation of a long extracellular filament. It is an essential virulence factor in many pathogenic species, and is also a priming component in the formation of antibiotic-resistant biofilms. The flagellum consists of the export apparatus and stator in the cytosol; the basal body, spanning the bacterial membrane(s) and periplasm; and the hook-filament, that protrudes away from the bacterial surface. Assembly of the bacterial flagellum is initiated by the formation of the basal body MS ring region, constituted of multiple copies of the protein FliF. Here, I report an analysis of the FliF sequence from various bacterial species, demonstrating that its periplasmic region is composed of a domain homologuous to that of the type III secretion system proteins PrgK, and of a second globular domain that possesses a similar fold to that of the sporulation complex component SpoIIIAG. I also describe that Chlamydia possesses an unusual FliF protein, lacking part of the PrgK homology domain and the SpoIIIAG-like domain, and fused to FliG at its C-terminus. Finally, I have combined the sequence analysis of FliF with the EM map of the MS ring, to propose the first atomic model for the FliF oligomer. These results further emphasize the similarity between the flagellum, T3SS and sporulation complex, and will facilitate further structural studies.

Biochemistry↗

bioRxiv2015

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted Fo region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the Fo region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the Fo region support a Brownian ratchet mechanism for proton-translocation driven rotation in ATP synthases.

Biochemistry↗

bioRxiv2015

A Novel Method and Simple On-line Tool for Maximum Likelihood Calibration of Immunoblots and other Measurements that are Quantified in Batches

Experimental measurements require calibration to transform measured signals into physically meaningful values. The conventional approach has two steps: the experimenter deduces a conversion function using measurements on standards and then calibrates (or normalizes) measurements on unknown samples with this function. The deduction of the conversion function from only the standard measurements causes the results to be quite sensitive to experimental noise. It also implies that any data collected without reliable standards must be discarded. Here we show that a new \"1-step calibration method\" reduces these problems for the common situation in which samples are measured in batches, where a batch could be an immunoblot (Western blot), an enzyme-linked immunosorbent assay (ELISA), a sequence of spectra, or a microarray, provided that some sample measurements are replicated across multiple batches. The 1-step method computes all calibration results iteratively from all measurements. It returns the most probable values for the sample compositions under the assumptions of a statistical model, making them the maximum likelihood predictors. It is less sensitive to measurement error on standards and enables use of some batches that do not include standards. In direct comparison of both real and simulated immunoblot data, the 1-step method consistently exhibited smaller errors than the conventional \"2-step\" method. These results suggest that the 1-step method is likely to be most useful for cases where experimenters want to analyze existing data that are missing some standard measurements and where experimenters want to extract the best results possible from their data. Simple open source software for both methods is available for download or on-line use.\n\nAuthor SummaryMost quantitative measurements do not return the physical quantities that are of interest, but some instrument-specific response value instead. These measurements are then converted to physical quantities through a conversion function, which the experimenter deduces from instrument responses for one or more standard samples of known composition. This is called calibration or normalization. For example, we recently performed quantitative immunoblotting on a large number of samples, each replicated on multiple blots, and then calibrated the measurements to yield protein concentrations relative to those in a standard sample. We found that the conventional calibration approach of treating the samples in each blot independently of the samples in other blots produced inaccurate results because this approach is completely dependent on the standard measurements, which were sometimes missing or erroneous in our data. Thus, we developed a new calibration approach in which we fit a statistical model to the entire data set simultaneously. This method, which applies to a very wide range of calibration problems, was substantially more accurate during validation tests and can be shown to return the most accurate results possible within the assumptions of the model. It is particularly useful when some standard measurements are missing from data sets or when experimenters want the best possible results.

Biochemistry↗

bioRxiv2015

Mechanism-based sirtuin enzyme activation

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.

Biochemistry↗

bioRxiv2015

Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

Pseudomonas aeruginosa is a major cause of chronic infections in cystic fibrosis patients. The nucleotidyl cyclase toxin ExoY is a virulence factors injected by the pathogen and associated with severe damage to lung tissue. ExoY-like cyclases are also found in other Gram-negative pathogens and shown to contribute to virulence, although they remained poorly characterized. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown co-factor that activates P. aeruginosa ExoY within host target cells. Highly purified actin, when polymerized into filaments, potently stimulates (>10,000 fold) ExoY activity. ExoY co-localizes in vivo with actin filaments in transfected cells and, in vitro, it interferes with the regulation of actin assembly/disassembly-dynamics mediated by important F-actin-binding proteins. We further show that actin also activates an ExoY-like adenylate cyclase from a Vibrio species. Our results thus highlight a new sub-class within the class II adenylyl cyclase family, defined as actin-activated nucleotidyl cyclase (AA-NC) toxins.

Biochemistry↗

bioRxiv2015

Induced-fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids

The catalytic site of most enzymes can essentially deal with only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the inducedfit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. It allows us to highlight the rationale of the PTC structural organization, which confers to the ribosome the very unusual ability to handle large as well as small substrates.

Biochemistry↗

bioRxiv2015

Activation of G proteins by guanine nucleotide exchange factors relies on GTPase activity

G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an activation/inactivation cycle. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity - emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a balance/imbalance mechanism. This result has implications for the understanding of many intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems.

Biochemistry↗

bioRxiv2015

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins

The clustered regularly interspaced short palindromic repeat (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed \"in vitro enChIP\" using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first demonstrate that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we show that this technology can be employed to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis.

Biochemistry↗

bioRxiv2015

Modeling error in experimental assays using the bootstrap principle: Understanding discrepancies between assays using different dispensing technologies

All experimental assay data contains error, but the magnitude, type, and primary origin of this error is often not obvious. Here, we describe a simple set of assay modeling techniques based on the bootstrap principle that allow sources of error and bias to be simulated and propagated into assay results. We demonstrate how deceptively simple operations--such as the creation of a dilution series with a robotic liquid handler--can significantly amplify imprecision and even contribute substantially to bias. To illustrate these techniques, we review an example of how the choice of dispensing technology can impact assay measurements, and show how large contributions to discrepancies between assays can be easily understood and potentially corrected for. These simple modeling techniques--illustrated with an accompanying IPython notebook--can allow modelers to understand the expected error and bias in experimental datasets, and even help experimentalists design assays to more effectively reach accuracy and imprecision goals.

Biochemistry↗

bioRxiv2015

Characterizing RNA structures in vitro and in vivo with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

RNA molecules adopt a wide variety of structures that perform many cellular functions, including catalysis, small molecule sensing, and cellular defense, among others. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

Biochemistry↗

bioRxiv2015

Estimation of the lag time in a subsequent monomer addition model for fibril elongation

Fibrillogenesis, the production or development of protein fibers, has been linked to protein folding diseases. The progress curve of fibrils or aggregates typically takes on a sigmoidal shape witha lag phase, a rapid growth phase, and a final plateau regime. The study of the lag phase and the estimation of its critical timescale provide insight into the factors regulating the fibrillation process. However, methods to estimate a quantitative expression for the lag time rely on empirical expressions, which cannot connect the lag time to kinetic parameters associated with the reaction mechanisms of protein fibrillation. Here we introduce an approach for the estimation of the lag time using the governing rate equations of the elementary reactions of a subsequent monomer addition model for protein fibrillation as a case study. We show that the lag time is given by the sum of the critical timescales for each fibril intermediate in the subsequent monomer addition mechanism and therefore reveals causal connectivity between intermediate species. Furthermore, we find that single-molecule assays of protein fibrillation can exhibit a lag phase without a nucleation process, while dyes and extrinsic fluorescent probe bulk assays of protein fibrillation do not exhibit an observable lag time phase during template-dependent elongation. Our approach could be valuable for investigating the effects of intrinsic and extrinsic factors to the protein fibrillation reaction mechanism and provides physicochemical insights into parameters regulating the lag phase.

Biochemistry↗

bioRxiv2016

Proteomic indicators of oxidation and hydration state in colorectal cancer

New integrative approaches are needed to harness the potential of rapidly growing datasets of protein expression and microbial community composition in colorectal cancer (CRC). Chemical and thermodynamic models offer theoretical tools to describe populations of biomacromolecules and their relative potential for formation in different microenvironmental conditions. The average oxidation state of carbon (ZC) can be calculated as an elemental ratio from the chemical formulas of proteins, and water demand per residue [Formula] is computed by writing the overall formation reactions of proteins from basis species. Using reported results from clinical proteomic studies and microbial community profiling, many datasets exhibit higher mean ZC or [Formula] of proteins in carcinoma or adenoma compared to normal tissue. Microbial protein compositions have lower ZC in bacteria enriched in fecal samples from cancer patients. In thermodynamic calculations, the potential for formation of the cancer-related proteins is energetically favored by changes in the chemical activity of H2O and fugacity of O2 that reflect the compositional differences. The compositional analysis suggests that a systematic shift in chemical composition is an essential feature of the cancer proteome, and the thermodynamic descriptions show that the observed proteomic transformations could be promoted by microenvironments shifted toward more oxidizing conditions and increased hydration levels.

Biochemistry↗

bioRxiv2016

RNA structure through multidimensional chemical mapping

The discoveries of myriad non-coding RNA molecules, each transiting through multiple flexible states in cells or virions, present major challenges for structure determination. Advances in high-throughput chemical mapping give new routes for characterizing entire transcriptomes in vivo, but the resulting one-dimensional data generally remain too information-poor to allow accurate de novo structure determination. Multidimensional chemical mapping (MCM) methods seek to address this challenge. Mutate-and-map (M2), RNA interaction groups by mutational profiling (RING-MaP and MaP-2D analysis) and multiplexed {middle dot}OH cleavage analysis (MOHCA) measure how the chemical reactivities of every nucleotide in an RNA molecule change in response to modifications at every other nucleotide. A growing body of in vitro blind tests and compensatory mutation/rescue experiments indicate that MCM methods give consistently accurate secondary structures and global tertiary structures for ribozymes, ribosomal domains and ligand-bound riboswitch aptamers up to two hundred nucleotides in length. Importantly, MCM analyses provide detailed information on structurally heterogeneous RNA states, such as ligand-free riboswitches, that are functionally important but difficult to resolve with other approaches. The sequencing requirements of currently available MCM protocols scale at least quadratically with RNA length, precluding general application to transcriptomes or viral genomes at present. We propose a modify-crosslink-map expansion to overcome this and other current limitations to resolving the in vivo RNA structurome.

Biochemistry↗

bioRxiv2016

Two-Dimensional NMR Lineshape Analysis

NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. However, applications of such analyses are frequently limited by peak overlap inherent to complex biomolecular systems. Moreover, systematic errors may arise due to the analysis of two-dimensional data using theoretical frameworks developed for one-dimensional experiments. Here we introduce a more accurate and convenient method for the analysis of such data, based on the direct quantum mechanical simulation and fitting of entire two-dimensional experiments, which we implement in a new software tool, TITAN (TITration ANalysis). We expect the approach, which we demonstrate for a variety of protein-protein and protein-ligand interactions, to be particularly useful in providing information on multi-step or multi-component interactions.

Biochemistry↗

bioRxiv2016

Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of microRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.

Biochemistry↗

bioRxiv2016

Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly

AbstractThe maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies.

Biochemistry↗

bioRxiv2016

Atomic structure of Hsp90:Cdc37:Cdk4 reveals Hsp90 regulates kinase via dramatic unfolding

The Hsp90 molecular chaperone and its Cdc37 co-chaperone help stabilize and activate over half of the human kinome. However, neither the mechanism by which these chaperones assist their client kinases nor why some kinases are addicted to Hsp90 while closely related family members are independent is known. Missing has been any structural understanding of these interactions, with no full-length structures of human Hsp90, Cdc37 or either of these proteins with a kinase. Here we report a 3.9[A] cryoEM structure of the Hsp90:Cdc37:Cdk4 kinase complex. Cdk4 is in a novel conformation, with its two lobes completely separated. Cdc37 mimics part of the kinase N-lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase {beta}5 strand and interacts with exposed N-and C-lobe interfaces, safely trapping the kinase in an unfolded state. Based on this novel structure and extensive previous data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.\n\nOne Sentence SummaryThe first structure of a chaperone:kinase complex reveals that the Hsp90 system modulates and stabilizes kinases via a functionally relevant, unfolded open state.

Biochemistry↗

bioRxiv2016

A Novel Initiation Pathway in Escherichia Coli Transcription

Initiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.\n\nSignificanceTranscription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

Biochemistry↗