EPR AND MOLECULAR MODELING STUDIES ON NITROXIDE -LABELED NUCLEIC ACIDS AND REVERSE TRANSCRIPTASE SYSTEMS
Two facets of nitroxides are examined in this thesis. First, the reproducibility of their experimental detection by EPR (Electron Paramagnetic Resonance) on two table top Bruker e-scans in view of their potential use for gene detection, and, second, their use for long range distance measurements by molecular modeling in complex biological systems. EPR signal heights as a function of microwave power showed non-linear behavior on organic radicals (5-and 6-membered nitroxides), and on an inorganic standard (Cr3+) under non-saturating conditions with two Bruker table top e-scans. h-1/ho ratio measurements for determining gene probe degradation gave values with a small standard deviation at 10-6 M [nitroxide], especially with the e-scan that features higher microwave power output and uses an internal standard to correct for magnetic field drifts. Therefore, EPR based gene detection will no longer require high-end EPR instrumentation. For nitroxide – nitroxide distance determinations various software and strategies were examined for bi-nitroxide-labeled DNA and nitroxide-labeled DNA – nitroxide-labeled reverse transcriptase (RT) complexes. Of particular interest was the effect of the tether property (rigid, semi-rigid, and flexible) on nitroxide – nitroxide distances. The distances determined by molecular modeling are discussed in the context of recent EPR measurements on bi-nitroxide-labeled DNAs and complexes consisting of reverse transcriptase (RT) and DNA with strategically placed nitroxides. WebLab ViewerPro, a simple visualization program, was used to calculate nitroxide – nitroxide distances with rigid and semi-flexible tethered nitroxides. This program approximated experimental distances within the experimental error limits of EPR data only with rigid tethered nitroxides known to perturb molecular structures. The biologically more relevant semi- or flexible tethered nitroxides that have been shown to cause minimal structural perturbation required more advanced programs, such as Insight 2000.1, for distance calculations. Three approaches were systematically explored with Insight 2000.1 and are listed in increasing order of their complexity: Manual rotation (MR), Energy minimization (EM), and Molecular Dynamics (MD). The calculated distances were found to be method dependent, especially with the more flexible tethered nitroxides. In addition, a non-linear increase of the nitroxide distances as a function of base pair (bp) separation was only observed with rigid and semi-rigid tethered nitroxides. MD distances compared most favorably with those determined by EPR, and polar plots (Wingz plots) for monitoring spatial restriction displayed the highest degree of nitroxide freedom with the most flexible tethered nitroxides. MD on RT systems showed drastic distance changes with nitroxides strategically placed in the finger and thumb sub-domain of RT when a DNA substrate was introduced in the polymerase binding pocket. The distances calculated were within the error limit of EPR data. In conclusion, an analysis of nitroxide – nitroxide distances by molecular modeling is the most rigorous with rigid tethered nitroxides, less so with semi-rigid tethered nitroxides, and the least with flexibly tethered nitroxide.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:molecular modeling epr nitroxide labels nucleic acids reverse trancriptases
Date of Publication:01/01/2007