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MOLECULAR AND COMPUTATIONAL ANALYSIS OF SPIN-LABELED
NUCLEIC ACIDS AND PROTEINS
by
Ma’mon M. Hatmal
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
May 2011
Copyright 2011 Ma’mon M. Hatmal
Object Description
| Title | Molecular and computational analysis of spin-labeled nucleic acids and proteins |
| Author | Hatmal, Ma'mon M. |
| Author email | hatmal@usc.edu; mamoon_hatmal@yahoo.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Biochemistry & Molecular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2011-01-14 |
| Date submitted | 2011 |
| Restricted until | Unrestricted |
| Date published | 2011-02-19 |
| Advisor (committee chair) | Haworth, Ian S. |
| Advisor (committee member) |
Tokes, Zoltan Langen, Ralf Lieber, Michael R. Qin, Peter |
| Abstract | The biological function of RNA and DNA molecules depends on their three-dimensional folds. Many biophysical methods have been used to determine structures of RNA and DNA, including X-ray crystallography and NMR spectroscopy. However, it is difficult to use these methods for complex folds. Pulse electron paramagnetic resonance (EPR) using the double electron electron resonance (DEER) sequence can be used also to measure distances between spin labels at two positions in nucleic acids, and this method offers an alternative approach to structure determination. However, conversion of DEER data into structures requires a computational approach. Prior to the work described in this thesis, an in-house algorithm, NASNOX, was available for addition of spin labels to DNA and RNA and calculation of inter-label distances for known structures. The goal of the work in the thesis was to develop and test new algorithms for prediction of structure based on DEER data, using inter-label distances calculated with NASNOX and other constraints.; In the first algorithm (JUNFOLD), point representations of spin-label distributions and translations and rotations of helical axes are used to produce simplified models of spin-labeled junction conformations that can be compared with experimental data. Simple RNA and DNA junctions were tested using 'experimental' data calculated with NASNOX, and good RMSD values were obtained for the predicted structures. This algorithm was then used for prediction of the structure of the packaging RNA (pRNA) dimer based on experimental DEER data. The derived structure fitted well with inter-label distances and biochemical data.; Despite the successful use of JUNFOLD, the long running time was a concern. Therefore, another algorithm (JUNFOLD-M) was developed based on solving a complex system of algebraic equations corresponding to inter-label distances using a quasi-Newton method, in which solutions represent the coordinates of the average points. This approach produced results similar to those of JUNFOLD, but with a running time that was 10 to 100 times faster.; The effect of backbone and sequence on inter-label distances and label behavior was examined in two ways. First, a library of inter-label distances and structural geometries were derived for DNA and RNA helices. These data can be used to determine the helix conformation based on inter-label distances. Second, molecular dynamics simulations were performed for DNA duplexes to obtain structural information on the labeled site, correlate this information with experimental EPR findings, and determine the effect of sequence, label diastereoisomer, and type of label on the EPR spectrum. These studies provide information that can be reduced into a simple empirical model for calculation of inter-label distances in NASNOX.; The ideas developed for DNA and RNA in the NASNOX, JUNFOLD and JUNFOLD-M algorithms are also applicable to protein structure. An algorithm, PRONOX, was developed for addition of labels at specific sites of a protein and calculation of inter-label distances (this algorithm is equivalent to NASNOX). PRONOX calculations for 44 DEER distances determined for 5 proteins gave a high correlation coefficient between the theoretical and experimental data, and PRONOX was implemented in JUNFOLD for use in protein structure determination.; The algorithms developed in this thesis provide a package of routines for conversion of DEER data into biomolecular structure. With further modification, they will also permit inclusion of other experimental data, and should be of importance in future determination of RNA and DNA folds and protein conformation. |
| Keyword | computational; structure prediction; electron paramagnetic resonance (EPR); site-directed spin-labeling (SDSL); double electron-electron resonance (DEER); proteins; nucleic acids |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m3666 |
| Rights | Hatmal, Ma'mon M. |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Hatmal-4301 |
| Archival file | uscthesesreloadpub_Volume44/etd-Hatmal-4301.pdf |
Description
| Title | Page 1 |
| Full text | MOLECULAR AND COMPUTATIONAL ANALYSIS OF SPIN-LABELED NUCLEIC ACIDS AND PROTEINS by Ma’mon M. Hatmal A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOCHEMISTRY AND MOLECULAR BIOLOGY) May 2011 Copyright 2011 Ma’mon M. Hatmal |
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