Publications by authors named "Sandra J Greive"

15 Publications

  • Page 1 of 1

Thermostable virus portal proteins as reprogrammable adapters for solid-state nanopore sensors.

Nat Commun 2018 11 7;9(1):4652. Epub 2018 Nov 7.

Department of Physics, Northeastern University, Boston, MA, 02115, USA.

Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules.
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http://dx.doi.org/10.1038/s41467-018-07116-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220183PMC
November 2018

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

ACS Nano 2017 12 15;11(12):11931-11945. Epub 2017 Nov 15.

York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function.
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http://dx.doi.org/10.1021/acsnano.7b06980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963890PMC
December 2017

Structure of the large terminase from a hyperthermophilic virus reveals a unique mechanism for oligomerization and ATP hydrolysis.

Nucleic Acids Res 2017 Dec;45(22):13029-13042

York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK.

The crystal structure of the large terminase from the Geobacillus stearothermophilus bacteriophage D6E shows a unique relative orientation of the N-terminal adenosine triphosphatase (ATPase) and C-terminal nuclease domains. This monomeric 'initiation' state with the two domains 'locked' together is stabilized via a conserved C-terminal arm, which may interact with the portal protein during motor assembly, as predicted for several bacteriophages. Further work supports the formation of an active oligomeric state: (i) AUC data demonstrate the presence of oligomers; (ii) mutational analysis reveals a trans-arginine finger, R158, indispensable for ATP hydrolysis; (iii) the location of this arginine is conserved with the HerA/FtsK ATPase superfamily; (iv) a molecular docking model of the pentamer is compatible with the location of the identified arginine finger. However, this pentameric model is structurally incompatible with the monomeric 'initiation' state and is supported by the observed increase in kcat of ATP hydrolysis, from 7.8 ± 0.1 min-1 to 457.7 ± 9.2 min-1 upon removal of the C-terminal nuclease domain. Taken together, these structural, biophysical and biochemical data suggest a model where transition from the 'initiation' state into a catalytically competent pentameric state, is accompanied by substantial domain rearrangements, triggered by the removal of the C-terminal arm from the ATPase active site.
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http://dx.doi.org/10.1093/nar/gkx947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727402PMC
December 2017

Chimeric 14-3-3 proteins for unraveling interactions with intrinsically disordered partners.

Sci Rep 2017 09 20;7(1):12014. Epub 2017 Sep 20.

York Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, United Kingdom.

In eukaryotes, several "hub" proteins integrate signals from different interacting partners that bind through intrinsically disordered regions. The 14-3-3 protein hub, which plays wide-ranging roles in cellular processes, has been linked to numerous human disorders and is a promising target for therapeutic intervention. Partner proteins usually bind via insertion of a phosphopeptide into an amphipathic groove of 14-3-3. Structural plasticity in the groove generates promiscuity allowing accommodation of hundreds of different partners. So far, accurate structural information has been derived for only a few 14-3-3 complexes with phosphopeptide-containing proteins and a variety of complexes with short synthetic peptides. To further advance structural studies, here we propose a novel approach based on fusing 14-3-3 proteins with the target partner peptide sequences. Such chimeric proteins are easy to design, express, purify and crystallize. Peptide attachment to the C terminus of 14-3-3 via an optimal linker allows its phosphorylation by protein kinase A during bacterial co-expression and subsequent binding at the amphipathic groove. Crystal structures of 14-3-3 chimeras with three different peptides provide detailed structural information on peptide-14-3-3 interactions. This simple but powerful approach, employing chimeric proteins, can reinvigorate studies of 14-3-3/phosphoprotein assemblies, including those with challenging low-affinity partners, and may facilitate the design of novel biosensors.
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http://dx.doi.org/10.1038/s41598-017-12214-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607241PMC
September 2017

Development of a multipurpose scaffold for the display of peptide loops.

Protein Eng Des Sel 2017 06;30(6):419-430

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK. Correspondence:

Protein-protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. The design of the RAD display system includes differently tagged versions of the scaffold, which allows for flexibility in the protein purification method, and chemical coupling for small molecule labeling or surface immobilization. When combined with the significant thermal stability of the RadA protein, these features create a versatile multipurpose scaffold system. Using various orthogonal biophysical techniques, we show that peptides displayed on the scaffold bind to their natural targets in a fashion similar to linear parent peptides. We use the examples of CK2β/CK2α kinase and TPX2/Aurora A kinase protein complexes to demonstrate that the peptide displayed by the RAD scaffold can be used in PPI studies with the same binding efficacy but at lower costs compared with their linear synthetic counterparts.
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http://dx.doi.org/10.1093/protein/gzx017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897841PMC
June 2017

Viral genome packaging terminase cleaves DNA using the canonical RuvC-like two-metal catalysis mechanism.

Nucleic Acids Res 2017 04;45(6):3580-3590

York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK.

Bacteriophages and large dsDNA viruses encode sophisticated machinery to translocate their DNA into a preformed empty capsid. An essential part of this machine, the large terminase protein, processes viral DNA into constituent units utilizing its nuclease activity. Crystal structures of the large terminase nuclease from the thermophilic bacteriophage G20c show that it is most similar to the RuvC family of the RNase H-like endonucleases. Like RuvC proteins, the nuclease requires either Mn2+, Mg2+ or Co2+ ions for activity, but is inactive with Zn2+ and Ca2+. High resolution crystal structures of complexes with different metals reveal that in the absence of DNA, only one catalytic metal ion is accommodated in the active site. Binding of the second metal ion may be facilitated by conformational variability, which enables the two catalytic aspartic acids to be brought closer to each other. Structural comparison indicates that in common with the RuvC family, the location of the two catalytic metals differs from other members of the RNase H family. In contrast to a recently proposed mechanism, the available data do not support binding of the two metals at an ultra-short interatomic distance. Thus we postulate that viral terminases cleave DNA by the canonical RuvC-like mechanism.
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http://dx.doi.org/10.1093/nar/gkw1354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389553PMC
April 2017

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

Biochemistry 2016 09 1;55(36):5021-7. Epub 2016 Sep 1.

York Structural Biology Laboratory, Department of Chemistry, University of York , York YO10 5DD, United Kingdom.

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.
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http://dx.doi.org/10.1021/acs.biochem.6b00682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5193468PMC
September 2016

DNA recognition for virus assembly through multiple sequence-independent interactions with a helix-turn-helix motif.

Nucleic Acids Res 2016 Jan 15;44(2):776-89. Epub 2015 Dec 15.

York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK

The helix-turn-helix (HTH) motif features frequently in protein DNA-binding assemblies. Viral pac site-targeting small terminase proteins possess an unusual architecture in which the HTH motifs are displayed in a ring, distinct from the classical HTH dimer. Here we investigate how such a circular array of HTH motifs enables specific recognition of the viral genome for initiation of DNA packaging during virus assembly. We found, by surface plasmon resonance and analytical ultracentrifugation, that individual HTH motifs of the Bacillus phage SF6 small terminase bind the packaging regions of SF6 and related SPP1 genome weakly, with little local sequence specificity. Nuclear magnetic resonance chemical shift perturbation studies with an arbitrary single-site substrate suggest that the HTH motif contacts DNA similarly to how certain HTH proteins contact DNA non-specifically. Our observations support a model where specificity is generated through conformational selection of an intrinsically bent DNA segment by a ring of HTHs which bind weakly but cooperatively. Such a system would enable viral gene regulation and control of the viral life cycle, with a minimal genome, conferring a major evolutionary advantage for SPP1-like viruses.
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http://dx.doi.org/10.1093/nar/gkv1467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737164PMC
January 2016

Investigation of DNA sequence recognition by a streptomycete MarR family transcriptional regulator through surface plasmon resonance and X-ray crystallography.

Nucleic Acids Res 2013 Aug 7;41(14):7009-22. Epub 2013 Jun 7.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Consistent with their complex lifestyles and rich secondary metabolite profiles, the genomes of streptomycetes encode a plethora of transcription factors, the vast majority of which are uncharacterized. Herein, we use Surface Plasmon Resonance (SPR) to identify and delineate putative operator sites for SCO3205, a MarR family transcriptional regulator from Streptomyces coelicolor that is well represented in sequenced actinomycete genomes. In particular, we use a novel SPR footprinting approach that exploits indirect ligand capture to vastly extend the lifetime of a standard streptavidin SPR chip. We define two operator sites upstream of sco3205 and a pseudopalindromic consensus sequence derived from these enables further potential operator sites to be identified in the S. coelicolor genome. We evaluate each of these through SPR and test the importance of the conserved bases within the consensus sequence. Informed by these results, we determine the crystal structure of a SCO3205-DNA complex at 2.8 Å resolution, enabling molecular level rationalization of the SPR data. Taken together, our observations support a DNA recognition mechanism involving both direct and indirect sequence readout.
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http://dx.doi.org/10.1093/nar/gkt523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737563PMC
August 2013

The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

J Biol Chem 2013 Feb 28;288(7):5149-56. Epub 2012 Dec 28.

Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.
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http://dx.doi.org/10.1074/jbc.M112.419069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3576119PMC
February 2013

Fitting experimental transcription data with a comprehensive template-dependent modular kinetic model.

Biophys J 2011 Sep;101(5):1166-74

Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon, USA.

In the companion article, we developed a modular scheme for representing the kinetics of transcription elongation by RNA polymerase. As an example of how to use these approaches, in this article we use a comprehensive modular model of this sort to fit experimental transcript elongation results obtained on the canonical tR2 template of phage λ by means of complementary bulk gel electrophoresis and surface plasmon resonance assays. The gel electrophoresis results, obtained in experiments quenched at various times after initiation of transcription, provide distributions of RNA lengths as a function of time. The surface plasmon resonance methods were used to monitor increases and decreases in the total mass of transcription elongation complexes in the same experiments. The different measures of transcription dynamics that these methods provide allow us to use them in combination to obtain a set of largely robust and well-defined kinetic parameters. The results show that our modular approach can be used to develop and test predictive kinetic schemes that can be fit to real transcription elongation data. They also suggest that these approaches can be extended to simulate the kinetics of other processes that involve the processive extension or shortening of nucleic acid chains and related systems of sequential branching reaction events.
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http://dx.doi.org/10.1016/j.bpj.2011.07.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164171PMC
September 2011

Development of a "modular" scheme to describe the kinetics of transcript elongation by RNA polymerase.

Biophys J 2011 Sep;101(5):1155-65

Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon, USA.

Transcript elongation by RNA polymerase involves the sequential appearance of several alternative and off-pathway states of the transcript elongation complex (TEC), and this complicates modeling of the kinetics of the transcription elongation process. Based on solutions of the chemical master equation for such transcription systems as a function of time, we here develop a modular scheme for simulating such kinetic transcription data. This scheme deals explicitly with the problem of TEC desynchronization as transcript synthesis proceeds, and develops kinetic modules to permit the various alternative states of the TECs (paused states, backtracked states, arrested states, and terminated states) to be introduced one-by-one as needed. In this way, we can set up a comprehensive kinetic model of appropriate complexity to fit the known transcriptional properties of any given DNA template and set of experimental conditions, including regulatory cofactors. In the companion article, this modular scheme is successfully used to model kinetic transcription elongation data obtained by bulk-gel electrophoresis quenching procedures and real-time surface plasmon resonance methods from a template of known sequence that contains defined pause, stall, and termination sites.
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http://dx.doi.org/10.1016/j.bpj.2011.07.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164141PMC
September 2011

Monitoring RNA transcription in real time by using surface plasmon resonance.

Proc Natl Acad Sci U S A 2008 Mar 25;105(9):3315-20. Epub 2008 Feb 25.

Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.

The decision to elongate or terminate the RNA chain at specific DNA template positions during transcription is kinetically regulated, but the methods used to measure the rates of these processes have not been sufficiently quantitative to permit detailed mechanistic analysis of the steps involved. Here, we use surface plasmon resonance (SPR) technology to monitor RNA transcription by Escherichia coli RNA polymerase (RNAP) in solution and in real time. We show that binding of RNAP to immobilized DNA templates to form active initiation or elongation complexes can be resolved and monitored by this method, and that changes during transcription that involve the gain or loss of bound mass, including the release of the sigma factor during the initiation-elongation transition, the synthesis of the RNA transcript, and the release of core RNAP and nascent RNA at intrinsic terminators, can all be observed. The SPR method also permits the discrimination of released termination products from paused and other intermediate complexes at terminators. We have used this approach to show that the rate constant for transcript release at intrinsic terminators tR2 and tR' is approximately 2-3 s(-1) and that the extent of release at these terminators is consistent with known termination efficiencies. Simulation techniques have been used to fit the measured parameters to a simple kinetic model of transcription and the implications of these results for transcriptional regulation are discussed.
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http://dx.doi.org/10.1073/pnas.0712074105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2265150PMC
March 2008

Assembly of an RNA-protein complex. Binding of NusB and NusE (S10) proteins to boxA RNA nucleates the formation of the antitermination complex involved in controlling rRNA transcription in Escherichia coli.

J Biol Chem 2005 Oct 18;280(43):36397-408. Epub 2005 Aug 18.

Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403, USA.

Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K(d) values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K(d) values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available single-stranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.
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http://dx.doi.org/10.1074/jbc.M507146200DOI Listing
October 2005

Thinking quantitatively about transcriptional regulation.

Nat Rev Mol Cell Biol 2005 Mar;6(3):221-32

Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403, USA.

By thinking about the chemical and physical mechanisms that are involved in the stepwise elongation of RNA transcripts, we can begin to understand the way that these mechanisms are controlled within the cell to reflect the different requirements for transcription that are posed by various metabolic, developmental and disease states. Here, we focus on the mechanistic details of the single-nucleotide addition (or excision) cycle in the transcription process, as this is the level at which many regulatory mechanisms function and can be explained in quantitative terms.
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http://dx.doi.org/10.1038/nrm1588DOI Listing
March 2005