Michelle M. Spiering

Bio: Michelle M. Spiering is an academic researcher from Pennsylvania State University. The author has contributed to research in topics: Replisome & DNA replication. The author has an hindex of 19, co-authored 29 publications receiving 1191 citations. Previous affiliations of Michelle M. Spiering include University of Michigan & Centre national de la recherche scientifique.

Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase.

Abstract: Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-l-biopterin (H4B). Isolation of active enz...

Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism

Abstract: Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork (≈400 bp/s), its unwinding rate extrapolated to zero force is much slower (≈30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.

Substrate-driven chemotactic assembly in an enzyme cascade.

Abstract: Enzymes that form a metabolic pathway in which the product of one enzyme is the substrate for the next have now been shown to associate through a process of sequential, directed chemotactic movement. The extent of enzyme migration is proportional to the exposure time to the substrate gradient.

DNA polymerase as a molecular motor and pump.

Abstract: DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using fluorescence correlation spectroscopy, we demonstrate that the diffusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into the growing DNA template. The diffusion coefficient of the complex also shows a strong dependence on its inorganic cofactor, Mg2+ ions. When exposed to gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered fluid pumps. These miniature pumps are capable of transporting fluid and tracer particles in a directional manner with the pumping speed increasing in the presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature fluid ...

Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome

Abstract: The unwinding and priming activities of the bacteriophage T4 primosome, which consists of a hexameric helicase (gp41) translocating 5' to 3' and an oligomeric primase (gp61) synthesizing primers 5' to 3', have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a new DNA looping mechanism. A fused gp61-gp41 primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms by disfavoring primosome disassembly, thereby increasing primase processivity. In contrast to T4, priming in bacteriophage T7 and Escherichia coli involves discrete pausing of the primosome and dissociation of the primase from the helicase, respectively. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.

Nitric oxide synthases: structure, function and inhibition

Abstract: This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

Abstract: Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. Here we describe these techniques and illustrate them with examples highlighting current capabilities and limitations.

The ClusPro web server for protein-protein docking.

Abstract: The ClusPro server (https://cluspro.org) is a widely used tool for protein-protein docking. The server provides a simple home page for basic use, requiring only two files in Protein Data Bank (PDB) format. However, ClusPro also offers a number of advanced options to modify the search; these include the removal of unstructured protein regions, application of attraction or repulsion, accounting for pairwise distance restraints, construction of homo-multimers, consideration of small-angle X-ray scattering (SAXS) data, and location of heparin-binding sites. Six different energy functions can be used, depending on the type of protein. Docking with each energy parameter set results in ten models defined by centers of highly populated clusters of low-energy docked structures. This protocol describes the use of the various options, the construction of auxiliary restraints files, the selection of the energy parameters, and the analysis of the results. Although the server is heavily used, runs are generally completed in <4 h.

Conflict of interest statement. None declared.

Abstract: Hypertension 66 (20.3%) 24 (24.2%) 30 (16.3%) NS Diabetes 20 (6.2%) 7 (7.1%) 10 (5.4%) NS Excess weight 78 (24%) 27 (27.3%) 44 (23.9%) NS Smokers 64 (19.7%) 17 (17.2%) 35 (19.0%) NS Age >50 years 137 (42.2%) 54 (54.5%) 67 (36.4%) <0.02 Kidney disease 7 (2.2%) 1 (1%) 5 (2.7%) NS Family history, DM 102 (31.4%) 28 (28.3%) 66 (35.9%) NS