Publications by authors named "Borries Demeler"

140 Publications

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism.

Nat Commun 2021 03 31;12(1):1988. Epub 2021 Mar 31.

Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.
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http://dx.doi.org/10.1038/s41467-021-22253-6DOI Listing
March 2021

Density Matching Multi-wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations.

ACS Nano 2021 03 22;15(3):5068-5076. Epub 2021 Feb 22.

Department of Chemistry and Biochemistry, The University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.

Previous work suggested that lipid nanoparticle (LNP) formulations, encapsulating nucleic acids, display electron-dense morphology when examined by cryogenic-transmission electron microscopy (cryo-TEM). Critically, the employed cryo-TEM method cannot differentiate between loaded and empty LNP formulations. Clinically relevant formulations contain high lipid-to-nucleic acid ratios (10-25 (w/w)), and for systems that contain mRNA or DNA, it is anticipated that a substantial fraction of the LNP population does not contain a payload. Here, we present a method based on the global analysis of multi-wavelength sedimentation velocity analytical ultracentrifugation, using density matching with heavy water, that not only measures the standard sedimentation and diffusion coefficient distributions of LNP mixtures, but also reports the corresponding partial specific volume distributions and optically separates signal contributions from nucleic acid cargo and lipid shell. This makes it possible to reliably predict molar mass and anisotropy distributions, in particular, for systems that are heterogeneous in partial specific volume and have low to intermediate densities. Our method makes it possible to unambiguously measure the density of nanoparticles and is motivated by the need to characterize the extent to which lipid nanoparticles are loaded with nucleic acid cargoes. Since the densities of nucleic acids and lipids substantially differ, the measured density is directly proportional to the loading of nanoparticles. Hence, different loading levels will produce particles with variable density and partial specific volume. An UltraScan software module was developed to implement this approach for routine analysis.
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http://dx.doi.org/10.1021/acsnano.0c10069DOI Listing
March 2021

The BRPF1 bromodomain is a molecular reader of di-acetyllysine.

Curr Res Struct Biol 2020 12;2:104-115. Epub 2020 May 12.

Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, VT, 05446, USA.

Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex, which is associated with chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of chromatin reader domains including two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) domain. BRPF1 is known to recruit the MOZ HAT complex to chromatin by recognizing acetylated lysine residues on the N-terminal histone tail region through its bromodomain. However, histone proteins can contain several acetylation modifications on their N-terminus, and it is unknown how additional marks influence bromodomain recruitment to chromatin. Here, we identify the BRPF1 bromodomain as a selective reader of di-acetyllysine modifications on histone H4. We used ITC assays to characterize the binding of di-acetylated histone ligands to the BRPF1 bromodomain and found that the domain binds preferentially to histone peptides H4K5acK8ac and H4K5acK12ac. Analytical ultracentrifugation (AUC) experiments revealed that the monomeric state of the BRPF1 bromodomain coordinates di-acetylated histone ligands. NMR chemical shift perturbation studies, along with binding and mutational analyses, revealed non-canonical regions of the bromodomain-binding pocket that are important for histone tail recognition. Together, our findings provide critical information on how the combinatorial action of post-translational modifications can modulate BRPF1 bromodomain binding and specificity.
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http://dx.doi.org/10.1016/j.crstbi.2020.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7861561PMC
May 2020

Nucleic acid binding by SAMHD1 contributes to the antiretroviral activity and is enhanced by the GpsN modification.

Nat Commun 2021 02 2;12(1):731. Epub 2021 Feb 2.

Department of Biochemistry and Structural Biology, UT Health San Antonio, San Antonio, TX, USA.

SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3' to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.
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http://dx.doi.org/10.1038/s41467-021-21023-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854603PMC
February 2021

Human DDX17 Unwinds Rift Valley Fever Virus Non-Coding RNAs.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada.

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5' non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17) and RVFV non-coding RNAs, IGR and 5' NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17, IGR, and 5' NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5' NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17 can unwind both RNAs. Overall, our study provides direct evidence of DDX17 interacting with and unwinding RVFV non-coding regions.
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http://dx.doi.org/10.3390/ijms22010054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793125PMC
December 2020

Characterizing Drug-Polymer Interactions in Aqueous Solution with Analytical Ultracentrifugation.

Mol Pharm 2021 01 2;18(1):246-256. Epub 2020 Dec 2.

Department of Pharmaceutics, University of Minnesota, 308 Harvard St SE, Minneapolis, Minnesota 55455, United States.

We present a new approach for characterizing drug-polymer interactions in aqueous media, using sedimentation velocity analytical ultracentrifugation (AUC). We investigated the potential interaction of ketoconazole (KTZ), a poorly water-soluble drug, with polyacrylic acid (PAA) and a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) in aqueous buffers. The effect of the polymer on the sedimentation coefficient of the drug was the observable metric. The drug alone, when subjected to AUC, exhibited a very narrow sedimentation peak at 0.2 Svedberg (S), in agreement with the expectation for a monomeric drug with a molar mass < 1000 Dalton. Conversely, the neat polymers showed broad profiles with higher sedimentation coefficients, reflecting their larger more heterogeneous size distributions. The sedimentation profiles of the drug-polymer mixtures were expectedly different from the profile of the neat drug. With KTZ-Soluplus, a complete shift to faster sedimentation times (indicative of an interaction) was observed, while with KTZ-PAA, a split peak indicated the existence of the drug in both free and interacting states. The sedimentation profile of carbamazepine, a second model drug, in the presence of hydroxypropyl methyl cellulose acetate succinate (HPMCAS, another polymer) revealed multiple "populations" of drug-polymer species, very similar to the sedimentation profile of neat HPMCAS. The interactions probed by AUC were compared with the results from isothermal titration calorimetry. In vitro dissolution tests performed on amorphous solid dispersions prepared with the same drug-polymer pairs suggested that the interactions may play a role in prolonging drug supersaturation. The results show the possibility of characterizing drug-polymer interactions in aqueous solution with high hydrodynamic resolution, addressing a major challenge frequently encountered in the mechanistic investigations of the dissolution behavior of amorphous solid dispersions.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00849DOI Listing
January 2021

Measuring compressibility in the optima AUC™ analytical ultracentrifuge.

Eur Biophys J 2020 Dec 24;49(8):711-718. Epub 2020 Nov 24.

Department of Chemistry, University of Montana, Missoula, MT, USA.

A method is described to accurately measure the compressibility of liquids using an analytical ultracentrifuge. The method makes use of very large pressure gradients, which can be generated in the analytical ultracentrifuge at high speeds to induce a maximum compression signal. Taking advantage of the new Optima AUC, which offers 10 micron radial resolution, a novel calibration centerpiece for measuring rotor stretch, and a speed-ramping procedure, even the weak compressibility of liquids like water, typically considered to be incompressible, could be detected. A model using the standard expression for the secant-average bulk modulus describing the relative compression of a liquid in the analytical ultracentrifuge is derived. The model is a function of the loading volume and the hydrostatic pressure generated in the analytical ultracentrifuge, as well as the secant-average bulk modulus. The compressibility of water and toluene were measured and the linear secant-average bulk modulus and meniscus positions were fitted. In addition to the measurement of the compressibility of liquids, applications for this method include an improved prediction of boundary conditions for multi-speed analytical ultracentrifugation experiments to better describe highly heterogeneous systems with analytical speed-ramping procedures, and the prediction of radius-dependent density variations.
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http://dx.doi.org/10.1007/s00249-020-01482-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751350PMC
December 2020

Multi-wavelength analytical ultracentrifugation as a tool to characterise protein-DNA interactions in solution.

Eur Biophys J 2020 Dec 21;49(8):819-827. Epub 2020 Nov 21.

Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, PO Box 4800, Christchurch, 8140, New Zealand.

Understanding how proteins interact with DNA, and particularly the stoichiometry of a protein-DNA complex, is key information needed to elucidate the biological role of the interaction, e.g. transcriptional regulation. Here, we present an emerging analytical ultracentrifugation method that features multi-wavelength detection to characterise complex mixtures by deconvoluting the spectral signals of the interaction partners into separate sedimentation profiles. The spectral information obtained in this experiment provides direct access to the molar stoichiometry of the interacting system to complement traditional hydrodynamic information. We demonstrate this approach by characterising a multimeric assembly process between the transcriptional repressor of bacterial sialic acid metabolism, NanR and its DNA-binding sequence. The method introduced in this study can be extended to quantitatively analyse any complex interaction in solution, providing the interaction partners have different optical properties.
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http://dx.doi.org/10.1007/s00249-020-01481-6DOI Listing
December 2020

The pH-Induced Selectivity Between Cysteine or Histidine Coordinated Heme in an Artificial α-Helical Metalloprotein.

Angew Chem Int Ed Engl 2021 02 23;60(8):3974-3978. Epub 2020 Dec 23.

Laboratoire de Bioénergétique et Ingénierie des Protéines (UMR 7281), IMM FR3479, CNRS, Aix-Marseille Univ., Marseille, France.

De Novo metalloprotein design assesses the relationship between metal active site architecture and catalytic reactivity. Herein, we use an α-helical scaffold to control the iron coordination geometry when a heme cofactor is allowed to bind to either histidine or cysteine ligands, within a single artificial protein. Consequently, we uncovered a reversible pH-induced switch of the heme axial ligation within this simplified scaffold. Characterization of the specific heme coordination modes was done by using UV/Vis and Electron Paramagnetic Resonance spectroscopies. The penta- or hexa-coordinate thiolate heme (9≤pH≤11) and the penta-coordinate imidazole heme (6≤pH≤8.5) reproduces well the heme ligation in chloroperoxidases or cyt P450 monooxygenases and peroxidases, respectively. The stability of heme coordination upon ferric/ferrous redox cycling is a crucial property of the construct. At basic pHs, the thiolate mini-heme protein can catalyze O reduction when adsorbed onto a pyrolytic graphite electrode.
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http://dx.doi.org/10.1002/anie.202012673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902473PMC
February 2021

Moving analytical ultracentrifugation software to a good manufacturing practices (GMP) environment.

PLoS Comput Biol 2020 06 19;16(6):e1007942. Epub 2020 Jun 19.

University of Montana, Dept. of Chemistry, Missoula, Montana, United States of America.

Recent advances in instrumentation have moved analytical ultracentrifugation (AUC) closer to a possible validation in a Good Manufacturing Practices (GMP) environment. In order for AUC to be validated for a GMP environment, stringent requirements need to be satisfied; analysis procedures must be evaluated for consistency and reproducibility, and GMP capable data acquisition software needs to be developed and validated. These requirements extend to multiple regulatory aspects, covering documentation of instrument hardware functionality, data handling and software for data acquisition and data analysis, process control, audit trails and automation. Here we review the requirements for GMP validation of data acquisition software and illustrate software solutions based on UltraScan that address these requirements as far as they relate to the operation and data handling in conjunction with the latest analytical ultracentrifuge, the Optima AUC by Beckman Coulter. The software targets the needs of regulatory agencies, where AUC plays a critical role in the solution-based characterization of biopolymers and macromolecular assemblies. Biopharmaceutical and regulatory agencies rely heavily on this technique for characterizations of pharmaceutical formulations, biosimilars, injectables, nanoparticles, and other soluble therapeutics. Because of its resolving power, AUC is a favorite application, despite the current lack of GMP validation. We believe that recent advances in standards, hardware, and software presented in this work manage to bridge this gap and allow AUC to be routinely used in a GMP environment. AUC has great potential to provide more detailed information, at higher resolution, and with greater confidence than other analytical techniques, and our software satisfies an urgent need for AUC operation in the GMP environment. The software, including documentation, are publicly available for free download from Github. The multi-platform software is licensed by the LGPL v.3 open source license and supports Windows, Mac and Linux platforms. Installation instructions and a mailing list are available from ultrascan.aucsolutions.com.
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http://dx.doi.org/10.1371/journal.pcbi.1007942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347214PMC
June 2020

Neuropathy-associated histidyl-tRNA synthetase variants attenuate protein synthesis in vitro and disrupt axon outgrowth in developing zebrafish.

FEBS J 2021 Jan 6;288(1):142-159. Epub 2020 Jul 6.

Department of Biochemistry, University of Vermont, Burlington, VT, USA.

Charcot-Marie-Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length-dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl-tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT-associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl-tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor-stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT-HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild-type HARS1, raising the possibility that CMT-HARS1 mutations cause disease through a dominant-negative mechanism. Overall, these findings support the hypothesis that CMT-HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction.
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http://dx.doi.org/10.1111/febs.15449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736457PMC
January 2021

A calibration disk for the correction of radial errors from chromatic aberration and rotor stretch in the Optima AUC™ analytical ultracentrifuge.

Eur Biophys J 2020 Dec 9;49(8):701-709. Epub 2020 May 9.

Department of Chemistry and Biochemistry, The University of Lethbridge, 4401 University Drive, Lethbridge, AB, T1K 3M4, Canada.

Experiments performed in the analytical ultracentrifuge (AUC) measure sedimentation and diffusion coefficients, as well as the partial concentration of colloidal mixtures of molecules in the solution phase. From this information, their abundance, size, molar mass, density and anisotropy can be determined. The accuracy with which these parameters can be determined depends in part on the accuracy of the radial position recordings and the boundary conditions used in the modeling of the AUC data. The AUC instrument can spin samples at speeds up to 60,000 rpm, generating forces approaching 300,000 g. Forces of this magnitude will stretch the titanium rotors used in the instrument, shifting the boundary conditions required to solve the flow equations used in the modeling of the AUC data. A second source of error is caused by the chromatic aberration resulting from imperfections in the UV-visible absorption optics. Both errors are larger than the optical resolution of currently available instrumentation. Here, we report software routines that correct these errors, aided by a new calibration disk which can be used in place of the counterbalance to provide a calibration reference for each experiment to verify proper operation of the AUC instrument. We describe laboratory methods and software routines in UltraScan that incorporate calibrations and corrections for the rotor stretch and chromatic aberration in order to support Good Manufacturing Practices for AUC data analysis.
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http://dx.doi.org/10.1007/s00249-020-01434-zDOI Listing
December 2020

Nanoscale Structure Determination of Murray Valley Encephalitis and Powassan Virus Non-Coding RNAs.

Viruses 2020 02 8;12(2). Epub 2020 Feb 8.

Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada.

Viral infections are responsible for numerous deaths worldwide. Flaviviruses, which contain RNA as their genetic material, are one of the most pathogenic families of viruses. There is an increasing amount of evidence suggesting that their 5' and 3' non-coding terminal regions are critical for their survival. Information on their structural features is essential to gain detailed insights into their functions and interactions with host proteins. In this study, the 5' and 3' terminal regions of Murray Valley encephalitis virus and Powassan virus were examined using biophysical and computational modeling methods. First, we used size exclusion chromatography and analytical ultracentrifuge methods to investigate the purity of transcribed RNAs. Next, we employed small-angle X-ray scattering techniques to study solution conformation and low-resolution structures of these RNAs, which suggest that the 3' terminal regions are highly extended as compared to the 5' terminal regions for both viruses. Using computational modeling tools, we reconstructed 3-dimensional structures of each RNA fragment and compared them with derived small-angle X-ray scattering low-resolution structures. This approach allowed us to reinforce that the 5' terminal regions adopt more dynamic structures compared to the mainly double-stranded structures of the 3' terminal regions.
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http://dx.doi.org/10.3390/v12020190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077200PMC
February 2020

Probing RNA-Protein Interactions and RNA Compaction by Sedimentation Velocity Analytical Ultracentrifugation.

Methods Mol Biol 2020 ;2113:281-317

Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, Canada.

Recent advances in multi-wavelength analytical ultracentrifugation (MWL-AUC) combine the power of an exquisitely sensitive hydrodynamic-based separation technique with the added dimension of spectral separation. This added dimension has opened up new doors to much improved characterization of multiple, interacting species in solution. When applied to structural investigations of RNA, MWL-AUC can precisely report on the hydrodynamic radius and the overall shape of an RNA molecule by enabling precise measurements of its sedimentation and diffusion coefficients and identify the stoichiometry of interacting components based on spectral decomposition. Information provided in this chapter will allow an investigator to design experiments for probing ion and/or protein-induced global conformational changes of an RNA molecule and exploit spectral differences between proteins and RNA to characterize their interactions in a physiological solution environment.
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http://dx.doi.org/10.1007/978-1-0716-0278-2_19DOI Listing
January 2021

Structure-Function Studies of the Bacillus subtilis Ric Proteins Identify the Fe-S Cluster-Ligating Residues and Their Roles in Development and RNA Processing.

mBio 2019 09 17;10(5). Epub 2019 Sep 17.

Public Health Research Center of New Jersey Medical School, Newark, New Jersey, USA

In , the RicA (YmcA), RicF (YlbF), and RicT (YaaT) proteins accelerate the phosphorylation of the transcription factor Spo0A, contributing to genetic competence, sporulation, and biofilm formation, and are also essential for the correct maturation of several protein-encoding and riboswitch RNAs. These proteins form a stable complex (RicAFT) that carries two [4Fe-4S] clusters. We show here that the complex is a 1:1:1 heterotrimer, and we present the X-ray crystal structures of a RicAF heterotetramer and of a RicA dimer. We also demonstrate that one of the Fe-S clusters (cluster 1) is ligated by cysteine residues donated exclusively by RicT and can be retained when the RicT monomer is purified by itself. Cluster 2 is ligated by C167 from RicT, by C134 and C146 located near the C terminus of RicF, and by C141 at the C terminus of RicA. These findings imply the following novel arrangement: adjacent RicT residues C166 and 167 ligate clusters 1 and 2, respectively, while cluster 2 is ligated by cysteine residues from RicT, RicA, and RicF. Thus, the two clusters must lie close to one another and at the interface of the RicAFT protomers. We also show that the cluster-ligating cysteine residues, and therefore most likely both Fe-S clusters, are essential for mRNA maturation, for the regulation of transcript stability, and for several Ric-associated developmental phenotypes, including competence for transformation, biofilm formation, and sporulation. Finally, we present evidence that RicAFT, RicAF, and RicA and the RicT monomer may play distinct regulatory roles The RicA, RicF, and RicT proteins are widely conserved among the firmicute bacteria and play multiple roles in Among the phenotypes associated with the inactivation of these proteins are the inability to be genetically transformed or to form biofilms, a decrease in sporulation frequency, and changes in the stability and maturation of multiple RNA species. Despite their importance, the molecular mechanisms of Ric protein activities have not been elucidated and the roles of the two iron-sulfur clusters on the complex of the three proteins are not understood. To unravel the mechanisms of Ric action, molecular characterization of the complex and of its constituent proteins is essential. This report represents a major step toward understanding the structures of the Ric proteins, the arrangement and roles of the Fe-S clusters, and the phenotypes associated with Ric mutations.
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http://dx.doi.org/10.1128/mBio.01841-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751060PMC
September 2019

Novel combinations of experimental and computational analysis tested on the binding of metalloprotoporphyrins to albumin.

Int J Biol Macromol 2019 Aug 10;134:445-457. Epub 2019 May 10.

Department of Physics and Astronomy, University of Texas at San Antonio, 78249 San Antonio, TX, United States of America. Electronic address:

The evidence that Human Serum Albumin (HSA) binds metal ions and organometallic compounds has generated interest in its physiological role as a metalloprotein and as a vehicle for synthetic biology applications (e.g., synthetic blood and solar energy conversion). HSA has been shown to bind metallo-porphyrins, however, the structural details of such interactions are available only for the HSA:heme complex. A typical challenge for studying the interaction of proteins with metalloporphyrins is the poor solubility of the ligands that affect the characterization the complexes. The manuscript shows that a combination of dialysis and centrifugation yields aqueous solutions that contain >90% HSA:porphyrin complexes and virtually eliminate aggregated ligands. The removal of aggregates increases the quality of the optical spectroscopy data which, in turn, yield more accurate binding constants (~0.1 and 2.1 × 10 M) and reveal FRET between Trp214 and the porphyrins. The Trp-porphyrin distance was estimated to be within the 28-34 Å range and was used to guide the search of binding sites through a novel feedback approach with docking simulations. Results suggest while some protoporphyrins (metal-free, Fe(III)PPIX and Mg(II)PPIX) bind HSA at the heme site, others (Zn(II)PPIX, Mn(III)PPIX and Sn(IV)PPIX) are more likely to bind the Cys34.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.05.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608576PMC
August 2019

A Proteomic Screen of Neuronal Cell-Surface Molecules Reveals IgLONs as Structurally Conserved Interaction Modules at the Synapse.

Structure 2019 06 4;27(6):893-906.e9. Epub 2019 Apr 4.

Child Health Institute of New Jersey, New Brunswick, NJ 08901, USA; Departments of Neuroscience and Cell Biology Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA; Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA; School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand. Electronic address:

In the developing brain, cell-surface proteins play crucial roles, but their protein-protein interaction network remains largely unknown. A proteomic screen identified 200 interactions, 89 of which were not previously published. Among these interactions, we find that the IgLONs, a family of five cell-surface neuronal proteins implicated in various human disorders, interact as homo- and heterodimers. We reveal their interaction patterns and report the dimeric crystal structures of Neurotrimin (NTRI), IgLON5, and the neuronal growth regulator 1 (NEGR1)/IgLON5 complex. We show that IgLONs maintain an extended conformation and that their dimerization occurs through the first Ig domain of each monomer and is Ca independent. Cell aggregation shows that NTRI and NEGR1 homo- and heterodimerize in trans. Taken together, we report 89 unpublished cell-surface ligand-receptor pairs and describe structural models of trans interactions of IgLONs, showing that their structures are compatible with a model of interaction across the synaptic cleft.
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http://dx.doi.org/10.1016/j.str.2019.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609445PMC
June 2019

Crystallographic Structures of IlvN·Val/Ile Complexes: Conformational Selectivity for Feedback Inhibition of Aceto Hydroxy Acid Synthases.

Biochemistry 2019 04 29;58(15):1992-2008. Epub 2019 Mar 29.

Molecular Biophysics Unit , Indian Institute of Science , Bangalore , Karnataka 560012 , India.

Conformational factors that predicate selectivity for valine or isoleucine binding to IlvN leading to the regulation of aceto hydroxy acid synthase I (AHAS I) of Escherichia coli have been determined for the first time from high-resolution (1.9-2.43 Å) crystal structures of IlvN·Val and IlvN·Ile complexes. The valine and isoleucine ligand binding pockets are located at the dimer interface. In the IlvN·Ile complex, among residues in the binding pocket, the side chain of Cys is 2-fold disordered (χ angles of gauche and trans). Only one conformation can be observed for the identical residue in the IlvN·Val complexes. In a reversal, the side chain of His, located at the surface of the protein, exhibits two conformations in the IlvN·Val complex. The concerted conformational switch in the side chains of Cys and His may play an important role in the regulation of the AHAS I holoenzyme activity. A significant result is the establishment of the subunit composition in the AHAS I holoenzyme by analytical ultracentrifugation. Solution nuclear magnetic resonance and analytical ultracentrifugation experiments have also provided important insights into the hydrodynamic properties of IlvN in the ligand-free and -bound states. The structural and biophysical data unequivocally establish the molecular basis for differential binding of the ligands to IlvN and a rationale for the resistance of IlvM to feedback inhibition by the branched-chain amino acids.
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http://dx.doi.org/10.1021/acs.biochem.9b00050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668035PMC
April 2019

Measuring molecular interactions in solution using multi-wavelength analytical ultracentrifugation: combining spectral analysis with hydrodynamics.

Authors:
Borries Demeler

Biochem (Lond) 2019 1;41(2):14-18. Epub 2019 Apr 1.

University of Lethbridge and the Canadian Center for Hydrodynamics in Lethbridge, Canada, and the University of Montana, USA.

In 1926, the Swedish scientist Theodor Svedberg was awarded the Nobel Prize in Chemistry for his work on a disperse system, and for studying the colloidal properties of proteins. This work was, to a large extent, made possible by his invention of a revolutionary tool, the analytical ultracentrifuge. These days, technological advances in hardware and computing have transformed the field of analytical ultracentrifugation (AUC) by enabling entirely new classes of experiments and modes of measurement unimaginable by Svedberg, making AUC once again an indispensable tool for modern biomedical research. In this article these advances and their impact on studies of interacting molecules will be discussed, with particular emphasis on a new method termed multi-wavelength analytical ultracentrifugation (MWL-AUC). Novel detectors allow us to add a second dimension to the separation of disperse and heterogeneous systems: in addition to the traditional hydrodynamic separation of colloidal mixtures, it is now possible to identify the sedimenting molecules by their spectral absorbance properties. The potential for this advance is significant for the study of a large range of systems. A further advance has occurred in data management and computational capabilities, opening doors to improved analysis methods, as well as direct networking with the instrument, facilitating data acquisition and data handling, and significant increases in data density from faster detectors with higher resolution capability.
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http://dx.doi.org/10.1042/bio04102014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500494PMC
April 2019

Structural and functional characterisation of the entry point to pyocyanin biosynthesis in defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass.

Biosci Rep 2018 10 17;38(5). Epub 2018 Oct 17.

Biomolecular Interaction Centre and School of Physical and Chemical Sciences, University of Canterbury, Christchurch 8041, New Zealand

In (), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d--heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by as part of the duplicated phenazine biosynthetic cluster, from (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α and α, relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from The results described in the present study support an expanded classification of the type II DAH7PSs as type II and type II based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism.
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http://dx.doi.org/10.1042/BSR20181605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435475PMC
October 2018

Repurposing Triphenylmethane Dyes to Bind to Trimers Derived from Aβ.

J Am Chem Soc 2018 09 6;140(37):11745-11754. Epub 2018 Sep 6.

Department of Chemistry , University of California Irvine , Irvine , California 92697-2025 , United States.

Soluble oligomers of the β-amyloid peptide, Aβ, are associated with the progression of Alzheimer's disease. Although many small molecules bind to these assemblies, the details of how these molecules interact with Aβ oligomers remain unknown. This paper reports that crystal violet, and other C3 symmetric triphenylmethane dyes, bind to C3 symmetric trimers derived from Aβ. Binding changes the color of the dyes from purple to blue, and causes them to fluoresce red when irradiated with green light. Job plot and analytical ultracentrifugation experiments reveal that two trimers complex with one dye molecule. Studies with several triphenylmethane dyes reveal that three N, N-dialkylamino substituents are required for complexation. Several mutant trimers, in which Phe, Phe, and Ile were mutated to cyclohexylalanine, valine, and cyclohexylglycine, were prepared to probe the triphenylmethane dye binding site. Size exclusion chromatography, SDS-PAGE, and X-ray crystallographic studies demonstrate that these mutations do not impact the structure or assembly of the triangular trimer. Fluorescence spectroscopy and analytical ultracentrifugation experiments reveal that the dye packs against an aromatic surface formed by the Phe side chains and is clasped by the Ile side chains. Docking and molecular modeling provide a working model of the complex in which the triphenylmethane dye is sandwiched between two triangular trimers. Collectively, these findings demonstrate that the X-ray crystallographic structures of triangular trimers derived from Aβ can be used to guide the discovery of ligands that bind to soluble oligomers derived from Aβ.
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http://dx.doi.org/10.1021/jacs.8b06568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339561PMC
September 2018

Functionality of Redox-Active Cysteines Is Required for Restriction of Retroviral Replication by SAMHD1.

Cell Rep 2018 07;24(4):815-823

Department of Biochemistry & Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229, USA. Electronic address:

SAMHD1 is a dNTP triphosphohydrolase (dNTPase) that impairs retroviral replication in a subset of non-cycling immune cells. Here we show that SAMHD1 is a redox-sensitive enzyme and identify three redox-active cysteines within the protein: C341, C350, and C522. The three cysteines reside near one another and the allosteric nucleotide binding site. Mutations C341S and C522S abolish the ability of SAMHD1 to restrict HIV replication, whereas the C350S mutant remains restriction competent. The C522S mutation makes the protein resistant to inhibition by hydrogen peroxide but has no effect on the tetramerization-dependent dNTPase activity of SAMHD1 in vitro or on the ability of SAMHD1 to deplete cellular dNTPs. Our results reveal that enzymatic activation of SAMHD1 via nucleotide-dependent tetramerization is not sufficient for the establishment of the antiviral state and that retroviral restriction depends on the ability of the protein to undergo redox transformations.
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http://dx.doi.org/10.1016/j.celrep.2018.06.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067006PMC
July 2018

Sequence Reversal Prevents Chain Collapse and Yields Heat-Sensitive Intrinsic Disorder.

Biophys J 2018 07;115(2):328-340

Department of Chemistry and Biochemistry, Texas State University, San Marcos, Texas. Electronic address:

Sequence patterns of charge, hydrophobicity, hydrogen bonding, and other amino acid physicochemical properties contribute to mechanisms of protein folding, but how sequence composition and patterns influence the conformational dynamics of the denatured state ensemble is not fully understood. To investigate structure-sequence relationships in the denatured state, we reversed the sequence of staphylococcal nuclease and characterized its structure, thermodynamic character, and hydrodynamic radius using circular dichroism spectroscopy, dynamic light scattering, analytical ultracentrifugation, and size-exclusion chromatography as a function of temperature. The macromolecular size of "Retro-nuclease" is highly expanded in solution with characteristics similar to biological intrinsically disordered proteins. In contradistinction to a disordered state, Retro-nuclease exhibits a broad sigmoid transition of its hydrodynamic dimensions as temperature is increased, indicating a thermodynamically controlled compaction. Counterintuitively, the magnitude of these temperature-induced hydrodynamic changes exceed that observed from thermal denaturation of folded unaltered staphylococcal nuclease. Undetectable by calorimetry and intrinsic tryptophan fluorescence, the lack of heat capacity or fluorescence changes throughout the thermal transition indicate canonical hydrophobic collapse did not drive the Retro-nuclease structural transitions. Temperature-dependent circular dichroism spectroscopy performed on Retro-nuclease and computer simulations correlate to temperature sensitivity in the intrinsic sampling of backbone conformations for polyproline II and α-helix. The experimental results indicate a role for sequence direction in mediating the collapse of the polypeptide chain, whereas the simulation trends illustrate the generality of the observed heat effects on disordered protein structure.
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http://dx.doi.org/10.1016/j.bpj.2018.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050754PMC
July 2018

Two-dimensional grid optimization for sedimentation velocity analysis in the analytical ultracentrifuge.

Eur Biophys J 2018 Oct 17;47(7):837-844. Epub 2018 May 17.

Department of Biochemistry and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3901, USA.

Sedimentation velocity experiments performed in the analytical ultracentrifuge are modeled using finite-element solutions of the Lamm equation. During modeling, three fundamental parameters are optimized: the sedimentation coefficients, the diffusion coefficients, and the partial concentrations of all solutes present in a mixture. A general modeling approach consists of fitting the partial concentrations of solutes defined in a two-dimensional grid of sedimentation and diffusion coefficient combinations that cover the range of possible solutes for a given mixture. An increasing number of grid points increase the resolution of the model produced by the subsequent analysis, with denser grids giving rise to a very large system of equations. Here, we evaluate the efficiency and resolution of several regular grids and show that traditionally defined grids tend to provide inadequate coverage in one region of the grid, while at the same time being computationally wasteful in other sections of the grid. We describe a rapid and systematic approach for generating efficient two-dimensional analysis grids that balance optimal information content and model resolution for a given signal-to-noise ratio with improved calculation efficiency. These findings are general and apply to one- and two-dimensional grids, although they no longer represent regular grids. We provide a recipe for an improved grid-point spacing in both directions which eliminates unnecessary points, while at the same time providing a more uniform resolution that can be scaled based on the stochastic noise in the experimental data.
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http://dx.doi.org/10.1007/s00249-018-1309-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158092PMC
October 2018

Multi-speed sedimentation velocity simulations with UltraScan-III.

Eur Biophys J 2018 Oct 10;47(7):815-823. Epub 2018 May 10.

Department of Biochemistry and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229, USA.

Recent developments in the UltraScan-III software make it possible to model multi-speed analytical ultracentrifugation sedimentation velocity experiments using finite-element solutions of the Lamm equation. Using simulated data, we demonstrate here how these innovations can be used to enhance the resolution of sedimentation velocity experiments when compared to single-speed experiments. Using heterogeneous systems covering as much as five orders of magnitude in molar mass and fivefold in anisotropy, we compare results from runs performed at multiple speeds to those obtained from single-speed experiments, fitted individually and analyzed globally over multiple speeds, and quantify resolution for sample heterogeneous in size and anisotropy. We also provide guidance on the design of multi-speed experiments and offer a program that can be used to deduce optimal spacing of rotor speeds and speed step durations when a few parameters from the experiment can be estimated. These include the meniscus position, the sedimentation coefficient of the largest species in a mixture, and a solute distribution. Our results show that errors observed in the determination of hydrodynamic parameters for system with great heterogeneity are markedly reduced when multi-speed analysis is employed.
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http://dx.doi.org/10.1007/s00249-018-1308-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158090PMC
October 2018

Tetrahedral () Closed-Shell Cluster of 29 Silver Atoms & 12 Lipoate Ligands, [Ag(R-α-LA)]: Antibacterial and Antifungal Activity.

ACS Appl Nano Mater 2018 Apr 26;1(4):1595-1602. Epub 2018 Mar 26.

Department of Physics & Astronomy, University of Texas, San Antonio, Texas 78249, United States.

Here we report on the identification and applications of an aqueous 29-atom silver cluster stabilized with 12 lipoate ligands, i.e. Ag(R-α-LA) or (29,12), wherein R-α-LA = R-α-lipoic acid, a natural dithiolate. Its uniformity is checked by HPLC-ESI-MS and analytical ultracentrifugation, which confirms its small dimension (~3 nm hydrodynamic diameter). For the first time, this cluster has been detected intact via electrospray ionization mass spectrometry, allowing one to confirm its composition, its [3-] charge-state, and the 8-electron shell configuration of its metallic silver core. Its electronic structure and bonding, including T-symmetry and profound chirality in the outer shell, have been analyzed by DFT quantum-chemical calculations, starting from the known structure of a nonaqueous homologue. The cluster is effective against Methicillin-Resistant bacteria (MRSA) at a minimum inhibitory concentration (MIC) of 0.6 mg-Ag/mL. A preformed fungal biofilm, impermeable to other antifungal agents, was also inhibited by aqueous solutions of this cluster, in a dose-response manner, with a half-maximal inhibitory concentration (IC) of 0.94 mg-Ag/mL. Scanning electron micrographs showed the post-treatment ultrastructural changes on both MRSA and C. that are characteristic of those displayed after treatment by larger silver nanoparticles.
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http://dx.doi.org/10.1021/acsanm.8b00069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523820PMC
April 2018

Multi-wavelength analytical ultracentrifugation of human serum albumin complexed with porphyrin.

Eur Biophys J 2018 Oct 19;47(7):789-797. Epub 2018 Apr 19.

Department of Biochemistry and Structural Biology, UTHSCSA, San Antonio, TX, USA.

The new Beckman Coulter Optima AUC instrument, which features multi-wavelength detection that couples the hydrodynamic separation of colloidal mixtures to spectral deconvolution of interacting and non-interacting solutes present in a mixture, was used to analyze the composition of human serum albumin (HSA) bound to metallo-protoporphyrin. We present new methods implemented in UltraScan that permit Optima AUC-derived multi-wavelength data to be spectrally decomposed in the same fashion as has been made possible for the Cölfen detector earlier. We demonstrate this approach by spectrally separating sedimentation velocity experimental data from mixtures of apo-HSA and HSA complexed to different metallo-protoporphyrins. We further demonstrate how multi-wavelength AUC can accurately recover percentages of metallo-protoporphyrin-bound HSA and apo-HSA from mixtures and how multi-wavelength AUC permits the calculation of molar extinction coefficients for porphyrins bound to HSA. The presented method has broad applicability to other complex systems where mixtures of molecules with different spectral properties need to be characterized.
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http://dx.doi.org/10.1007/s00249-018-1301-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158097PMC
October 2018

Multi-speed sedimentation velocity implementation in UltraScan-III.

Eur Biophys J 2018 Oct 2;47(7):825-835. Epub 2018 Apr 2.

University of Texas Health Science Center, San Antonio, USA.

A framework for the global analysis of multi-speed analytical ultracentrifugation sedimentation velocity experiments is presented. We discuss extensions to the adaptive space-time finite element fitting methods implemented in UltraScan-III to model sedimentation velocity experiments where a single run is performed at multiple rotor speeds, and describe extensions in the optimization routines used for fitting experimental data collected at arbitrary multi-speed profiles. Our implementation considers factors such as speed dependent rotor stretching, the resulting radial shifting of the finite element solution's boundary conditions, and changes in the associated time-invariant noise. We also address the calculation of acceleration rates and acceleration zones from existing radial acceleration and time records, as well as utilization of the time state object available at high temporal resolution from the new Beckman Optima AUC instrument. Analysis methods in UltraScan-III support unconstrained models that extract reliable information for both the sedimentation and the diffusion coefficients. These methods do not rely on any assumptions and allow for arbitrary variations in both sedimentation and diffusion transport. We have adapted these routines for the multi-speed case, and developed optimized and general grid based fitting methods to handle changes in the information content of the simulation matrix for different speed steps. New graphical simulation tools are presented that assist the investigator to estimate suitable grid metrics and evaluate information content based on edit profiles for individual experiments.
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http://dx.doi.org/10.1007/s00249-018-1297-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6676492PMC
October 2018

States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM.

Biophys Rev 2018 Apr 14;10(2):583-596. Epub 2017 Dec 14.

Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, IN, 47907, USA.

Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.
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http://dx.doi.org/10.1007/s12551-017-0372-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899731PMC
April 2018