Final Report Summary - PHAGE MAS SSNMR (Structural characterization of filamentous bacteriophage viruses by magic-angle spinning solid-state NMR spectroscopy)
Filamentous bacteriophages comprise a family of viruses that share a similar virion structure and life cycle. The virion is composed of a circular ssDNA wrapped by thousands of similar copies of a major coat protein and several different minor coat proteins in both ends. Due to the mass (tens of MegaDaltons) and dimensions (~7 nm in diameter, 600 – 2000 nm in length) of filamentous phages, structural models have been obtained primarily from fiber diffraction, and from static solid state NMR of aligned concentrated solutions. Yet, these models are incomplete, and vary even within individual virions. Also, some key questions regarding DNA structure, subunit-DNA interactions, microscopic polymorphism, and more, remain unclear or are in debate. Magic-angle spinning solid state NMR (MAS SSNMR) has been shown to be a complementary and successful method for studying the structure and dynamics of biological macromolecules, and is particularly suitable for non-crystalline / non-soluble systems . Examples are membrane proteins, protein aggregates and fibrils, viruses and more. Also, sample preparation for MAS SSNMR is more flexible, and measurements can be performed in conditions, which are either biologically relevant, or important for their biophysical characterization.
In this project we used magic-angle spinning solid-state NMR to study the class-I bacteriophages belonging to the Ff family (fd, M13) and the class-II phage Pf1. We obtained the complete chemical shift assignment of the three phages, analyzed their intact capsid and DNA structure, examined similarities and differences, and investigated their structural behavior under different capsid and DNA perturbants and under different temperatures. We progressed significantly towards full structure calculation of M13, which is currently underway.
Sample preparation and purification: Routine high-yield preparation, purification and biophysical characterization of M13 and fd viruses are now common in our lab and regular yields range 30-50 mg / L of culture. We managed to prepare both fully and 'check-board' enriched samples using either 13C6-D-glucose, 1,3- 13C glycerol and 2- 13C glycerol, all combined with complete 15N enrichment. All samples retain their biological infectivity prior and after NMR experiments. NMR experiments for assignment and conformation studies: We coded, tested and applied multi-dimensional MAS NMR experiments to assign and study the various phage systems. In particular, we employed 13C13C homonuclear correlation experiments, 15N13C 2D and 3D heteronuclear correlation experiments and 2D double-quantum/single-quantum experiments. Additional experiments involve through-bond transfer in the solid-state. These experiments provided us with sufficient resolution to fully assign the viruses' capsid protein. The chemical shifts for the capsid of fd have been deposited in the BMRB (Accession #17728). The Figure on the right summarizes the results of the fd study: The coat protein has a single unique structure. It is mostly helical (residues 6-47), its first 5 residues have a loop-like structure and are very mobile, and the DNA has a C2′-endo/C3′-exo sugar pucker. Our results indicated that the structure of the wild-type phage is very homogeneous despite the indication of all other methods (fiber diffraction, cryoEM, static solid-state NMR) that a Y21M mutation is required to obtain a homogeneous sample.
In this project we used magic-angle spinning solid-state NMR to study the class-I bacteriophages belonging to the Ff family (fd, M13) and the class-II phage Pf1. We obtained the complete chemical shift assignment of the three phages, analyzed their intact capsid and DNA structure, examined similarities and differences, and investigated their structural behavior under different capsid and DNA perturbants and under different temperatures. We progressed significantly towards full structure calculation of M13, which is currently underway.
Sample preparation and purification: Routine high-yield preparation, purification and biophysical characterization of M13 and fd viruses are now common in our lab and regular yields range 30-50 mg / L of culture. We managed to prepare both fully and 'check-board' enriched samples using either 13C6-D-glucose, 1,3- 13C glycerol and 2- 13C glycerol, all combined with complete 15N enrichment. All samples retain their biological infectivity prior and after NMR experiments. NMR experiments for assignment and conformation studies: We coded, tested and applied multi-dimensional MAS NMR experiments to assign and study the various phage systems. In particular, we employed 13C13C homonuclear correlation experiments, 15N13C 2D and 3D heteronuclear correlation experiments and 2D double-quantum/single-quantum experiments. Additional experiments involve through-bond transfer in the solid-state. These experiments provided us with sufficient resolution to fully assign the viruses' capsid protein. The chemical shifts for the capsid of fd have been deposited in the BMRB (Accession #17728). The Figure on the right summarizes the results of the fd study: The coat protein has a single unique structure. It is mostly helical (residues 6-47), its first 5 residues have a loop-like structure and are very mobile, and the DNA has a C2′-endo/C3′-exo sugar pucker. Our results indicated that the structure of the wild-type phage is very homogeneous despite the indication of all other methods (fiber diffraction, cryoEM, static solid-state NMR) that a Y21M mutation is required to obtain a homogeneous sample.
