15] Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions

Abstract

Publisher Summary This chapter describes the application of filamentous bacteriophage as a versatile tool for generating partially ordered solutions of nucleic acids and proteins. The Pfl phage system described in the chapter is an ideal method for obtaining a tunable degree of alignment of RNA and DNA oligomers and some proteins in solution, which then allows the measurement of homonuclear and heteronuclear dipolar coupling interactions. The Pfl filamentous phage system is ideally suited for obtaining a tunable degree of alignment in RNA and DNA molecules and acidic proteins for the observation of dipolar couplings. The phage particles are fully aligned at magnetic fields as low as 300 MHz and are stable indefinitely at a range of temperatures and buffer conditions. These dipolar couplings provide long-range structural information that is presently unavailable by standard solution nuclear magnetic resonance (NMR) techniques; therefore, in the future, dipolar coupling data may prove to be as indispensable as 1 H– 1 H nuclear Overhauser effect (NOE) in the solution structure determinations of macromolecules. This should be especially true for structure determinations of RNA and DNA oligomers where the low density of protons compared to proteins leads to fewer useful NOE distance constraints, and therefore generally less well-defined structures. In addition, the current solution NMR techniques are generally unable to determine global structural features, such as bending in nucleic acids, due because of the local nature of the NOE distance constraints.