The internal DNA of the Pf1 bacteriophage is known from its dimensions to be the most extended naturally occurring DNA. Understanding its conformation is critical to further insights about DNA stability and packing processes in Pf1 and similar filamentous phages, and is of broader interest to biophysical studies of DNA. Structural studies of the intact 36 MDa Pf1 bacteriophage by solid-state NMR have, from their inception, been remarkably ambitious undertakings due to the size of the system and its structural complexity. Assignment and structural characterization of the major coat protein have been aided by symmetry and abundance of signal, and have been remarkably successful. However, it is only with the advent of improvements in methodology that the DNA of Pf1 can be studied. Recent rapid advances in techniques such as dynamic nuclear polarization have greatly improved sensitivity and made solid-state NMR studies applicable to a broader range of biopolymers and biological assemblies. The first high-resolution NMR study of the Pf1 DNA is presented herein. Assignment of the 13C and 15N resonances of the DNA at the level of nucleotide type has revealed a number of unusual chemical shifts, at or beyond the edges of their respective ranges in available databases. These database comparisons, especially at key conformational reporter sites such as sugar C3' and C5', confirm important details of existing structural models, such as a C2'-endo/gauche sugar pucker, anti glycosidic angle, an overall lack of base pairing, and the presence of aromatic stacking. Specific protein-DNA contacts consistent with those predicted by models are also observed.Fragment-based ab initio chemical shift prediction methods are employed in efforts to derive additional information from the experimental chemical shifts. The Pf1 DNA is found to be most consistent with models of highly stretched P-DNA derived from DNA stretching experiments, in contrast to more conventional forms like A- or Z-DNA. Further, the goodness-of-fit of existing structural models as well as several novel models is assessed; it is found that one of the new models, "Hybrid/2XKM", created by combining recent highly refined DNA and coat protein models, best reproduces experimental chemical shift patterns, and should likely be used as a starting point for subsequent refinements. Similar methodology is applied to the selectivity filter of the S. lividans potassium ion channel KcsA, finding that changes in ion occupancy alone are insufficient to reproduce experimental chemical shift perturbations. Hydration is important to the environment of the Pf1 DNA, and to our ability to detect it. NMR investigation of water populations in Pf1 samples reveals that water is in contact with a number of buried protein residues and the internal DNA, making a strong case for the existence of a pool of "internal hydration water." Such a water population has great potential to further benefit solid-state NMR studies of the Pf1 bacteriophage. Also, a new tool to study, analyze, and predict the effects of crystal contacts on solid-state NMR spectra is presented, along with a discussion of isotopic labeling strategies to reduce spectral congestion and aid in the collection of structural restraints for complex biomolecular assemblies.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D81G0TFX |
| Date | January 2012 |
| Creators | Sergeyev, Ivan |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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