Magic-Angle Spinning NMR of a Class I Filamentous Bacteriophage Virus

Abstract

The fd bacteriophage is a filamentous virus that is widely used for bio- and nanotechnology applications ranging from phage display to battery materials. The possibility of obtaining a detailed description of its structural properties regardless of its state is therefore essential not only for understanding its physical arrangement and its bacterial infection process but also for many other applications. Here we present a study of the fd phage by magic-angle spinning solid-state NMR. While current structures rely on a Y21M mutant, experiments performed on a strain bearing a wild-type capsid report on high symmetry of the phage and lack of explicit subunit polymorphism. Chemical shift analysis confirmed that the coat protein mostly consists of a rigid right-handed curved α-helix (residues 6-47 of 50), preceded by a flexible loop-structured N-terminus. We were able to qualitatively assign the resonances belonging to the DNA, including the deoxyribose sugars and the thymine bases. These chemical shifts are consistent with base stacking and a C2'-endo/C3'-exo sugar pucker.

The NMR–Rosetta capsid model of M13 bacteriophage reveals a quadrupled hydrophobic packing epitope

“…Pure, intact, infectious M13 phage particles were obtained as described before (32,33) and detailed in SI Appendix. Sparsely labeled samples were grown using 15 NH 4 Cl as a nitrogen source and on either [1,3-13 C]-glycerol and NaH 12 CO 3 or on [2-13 C]-glycerol and NaH 13 CO 3 , as the sole carbon sources (37).…”

“…Integrated approaches can be used to resolve structures of large assemblies (28,29) and recently, the combination of MAS NMR data, cryo-EM, and Rosetta modeling resulted in a detailed atomic structure of the recombinant type III secretion system needle (30,31). We have previously performed MAS NMR studies on both wt fd and M13 in a precipitated form (32)(33)(34). Their chemical shifts pointed to a single…”

Solid-state NMR: An emerging technique in structural biology of self-assemblies

“…High-resolution information can be extracted from 13 C/ 15 N labeled DNA in phages, as demonstrated for Pf1 [60], T7 [239] and fd [240] phages. Recently, the Goldbourt laboratory succeeded in obtaining very high quality MAS ssNMR spectra of several phages [240][241][242]. The use of 13 C spin diluted M13 samples, based on [1,3-13 C]-and [2-13 C]-glycerol carbon sources, resulted in the collection of a set of 214 unambiguous distances, including 59 intersubunit restraints [25].…”

Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface

“…The purified PEG-precipitated phage samples are packed into MAS NMR rotors under fully hydrated conditions (5 mg of T7 phage sample in a 3.2 mm rotor and 10-15 mg of Ff phage in a 4 mm rotor) and tests have shown that the phages are kept viable even after many rounds of experiments. The detailed protocols for the preparation of enriched phage samples can be found elsewhere [37,40].…”

“…We describe the experimental approaches to NMR studies of capsid structure and dynamics, DNA packing and protein-DNA interactions, and discuss their outcomes. The results presented here are based on a series of publications from recent years [37][38][39][40][41][42][43][44][45] and some new insights into silver-doped fd phage particles. While this article discusses bacteriophage viruses, in recent years a variety of ssNMR techniques have been successfully applied to study structure and dynamics of HIV viral capsids [46,47] and most recently proton detected experiments combined with fast MAS were utilized to study measle-virus capsids [48], both of which have human hosts.…”