Sunday, November 24, 2024
11/24/2024
Summary/Abstract for requested administrative supplement to GM-15792-57. With this administrative supplement to our current NIGMS grant (GM-15792-57) we seek funds to purchase a much-needed replacement surface plasmon resonance (SPR) instrument that is important for our current DNA replication studies and for other related work of our group. Substantial parts of our ongoing research efforts are focused on achieving a detailed mechanistic understanding of the function and control of the sub-assemblies of the DNA replication complex of bacteriophage T4 by applying spectroscopic methods that site-specifically position fluorescent and optically active probes in the DNA framework of the replication complex at important protein binding sites. We use these labeled constructs to monitor – both with bulk (ensemble) solution measurements and at the single-molecule level – biologically significant local conformational changes at defined DNA positions that have been labeled with our spectroscopic probes. SPR is an important analytical tool for monitoring equilibrium and dynamic properties of protein-DNA complexes, including those in which the DNA components have been labeled with optical probes. We are pursuing two different fluorescent probe-labeling strategies for our studies on DNA and protein-DNA interactions. We use pairs of spectrally visible iCy3 dyes placed (internally) within the DNA backbones at defined positions to study the structure and dynamics of local DNA backbone conformations and conformational fluctuations, while pairs of fluorescent 6-MI base analogues, which have been substituted for natural guanine DNA bases, are used to obtain complementary information about the structure and dynamics of the bases and base-pairs at defined sites within our model DNA replication-fork constructs. SPR measurements provide important information to support our ensemble and single-molecule measurements of probe-labeled systems, in addition to providing important ‘control’ information about these systems in the absence of probes. The single-channel manual SPR instrument currently available in our laboratory (and – to or knowledge – the only SPR instrument at our university) is over 15 years old, very time consuming to run, and its major components are now beginning to fail. Furthermore, our present instrument is so outdated that the manufacturer no longer supports its maintenance and components, and replacement microfluidic SPR sample cell ‘cartridges’ are no longer available commercially. We will soon exhaust our dwindling supply of these cells, which will then put our current instrument completely ‘out of business.’ For the above reasons, we herewith request funds to purchase a replacement multi-channel automatized SPR instrument, which will take much less time to run and will greatly facilitate our ongoing spectroscopic experiments, as well as our other ongoing studies of protein-nucleic acid interactions. [We note that our laboratory has considerable experience with using SPR techniques for studying protein-nucleic acid interactions – see Greive et al., Proc. Nat. Acad. Sci., 105, 3315, (2008); Greive et al., Biophys. J., 101, 1155, (2011); and Greive et al., Biophys. J. 101, 1166, (2011)].
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