An Electron Spin Resonance Study of DNA Dynamics Using the Slowly Relaxing Local Structure Model

Abstract

A systematic analysis is presented of the ESR spectra from DNA oligomers of a range of sizes, including a polymer, spin-labeled with nitroxide moieties attached by different tethers. The complexity of the DNA dynamics is dealt with by the use of the general slowly relaxing local structure (SRLS) model, wherein the nitroxide moiety is reorienting in a restricted local environment, which itself is relaxing on a longer time scale. The slower motion describes the global tumbling of the DNA lattice, and the faster motion, the internal dynamics. In the present analysis, the correlation times for the axially symmetric global tumbling were those obtained from hydrodynamic theory, while the correlation times for the internal dynamics and the order parameter, which directly measures its restricted range of motion, were determined by nonlinear least-squares fits to the spectra. The principal result is the observation and characterization of two types of spectra from these labeled DNA systems. These two spectra represent components that differ from each other with respect to their local environments, one a highly restricted site yielding a large order parameter (0.61) and slower internal motions and the other a much less restricted site (with order parameter of 0.18) and faster internal motions. Whereas the one-atom tethered DUTA exhibits both sites (in roughly 9:1 ratio with the more restricted site more prevalent), the two-atom tethered DUMTA and five-atom tethered DUAT exhibit just the more restricted site, but the five-atom tethered DUAP exhibits only the less restricted site. (DUAP differs from DUAT and the others in having a less flexible tether.) It is suggested that the spin labels trapped in the highly restricted/slow motional site have a stronger interaction with the base than those in the other site. In general, the longer the tether, the faster are the correlation times for internal dynamics. For all tethers, it is found that the correlation time for internal motion perpendicular to the internal symmetry axis systematically becomes slower as the size of the oligomer increases. It is suggested that this may be a manifestation of collective modes of motion of the DNA. It is pointed out that the simpler models used in previous ESR studies are simplified cases of the more realistic SRLS model.

Domain Flexibility in Ligand-Free and Inhibitor-BoundEscherichia coliAdenylate Kinase Based on a Mode-Coupling Analysis of15N Spin Relaxation

“…The local coupling potential exerted by the protein surroundings tends to align the N-H bond vector along the principal axes of the ordering frame. In the case of axially symmetric (along Z M ) local ordering it is most simply given by (10)(11)(12):…”

“…AKeco is the only adenylate kinase for which crystal structures corresponding to the extreme stages of the catalytic cycle are available (17,21,22), with the closed form represented by AKeco*AP 5 A. The ATP phosphates are bound to the enzyme partly through the so-called P-loop GXXGXGK (residues [7][8][9][10][11][12][13]. This binding motif between proteins and nucleotides occurs in many proteins that bind nucleoside triphosphates, in all the nucleoside monophosphate kinases, and in the weakly homologous G-proteins (17).…”

Site-directed Spin Labeling Studies on Nucleic Acid Structure and Dynamics

“…In each system, the rotational diffusion frame is fixed with respect to the structure of the nitroxide, although its exact orientation may vary from system to system. Similarly, the director is fixed at the target macromolecule, such as the helical axis of a DNA duplex (101), in a system-dependent fashion. Simulations provide both a rate (τ) and an order parameter (S) to describe the partially ordered (restrictive) diffusive motion of the nitroxide with respect to the target macromolecule.…”

“…The MOMD program is most useful in crystalline and liquid crystalline systems, but may also be used to describe systems where the global tumbling of the macromolecule is slow with respect to the EPR timescale. The MOMD approach has been used to simulate SDSL spectra in RNA (31,102) and DNA (101).…”

“…The model describes the situation where the spin label is reorienting with respect to the parent macromolecule, whereas the macromolecule is slowly tumbling. This model has been applied to DNA duplexes (101).…”