Conformational dynamics of an intact virus: Order parameters for the coat protein of Pf1 bacteriophage

Abstract

This study has examined the atomic-level dynamics of the protein in the capsid of filamentous phage Pf1. This capsid consists of Ϸ7,300 small subunits of only 46 aa in a helical array around a highly extended, circular single-stranded DNA molecule of 7,349 nt. Measurements were made of site-specific, solid-state NMR order parameters, ͗S͘, the values which are dimensionless quantities between 0 (mobile) and 1 (static) that characterize the amplitudes of molecular bond angular motions that are faster than microseconds. It was found that the protein subunit backbone is very static, and of particular interest, it appears to be static at residues glycine 15 and glutamine 16 where it had been previously thought to be mobile. In contrast to the backbone, several side chains display large-amplitude angular motions. Side chains on the virion exterior that interact with solvent are highly mobile, but surprisingly, the side chains of residues arginine 44 and lysine 45 near the DNA deep in the interior of the virion are also highly mobile. The largeamplitude dynamic motion of these positively charged side chains in their interactions with the DNA were not previously expected. The results reveal a highly dynamic aspect of a DNA-protein interface within a virus.solid-state NMR ͉ motion ͉ side chain T he Pf1 bacteriophage is a 2-m-long filamentous virus that infects Pseudomonas aeruginosa strain K (1, 2). The virion consists of a circular single-stranded DNA genome of 7,349 nt (3) within a cylindrical capsid of Ϸ7,300 major coat protein subunits that have 1:1 stoichiometric interactions (4, 5) with nucleotides along the filament, as well as a small number of other proteins interacting with reverse turns of DNA at the ends. A variety of spectroscopic and diffraction studies of Pf1 have been carried out (5-19), including studies by solid-state NMR of the protein in the intact virion (16)(17)(18)(19), as in the present study, and by solution NMR of the isolated protein in detergent micelles (8,14) as a model of its structure in the membrane before virion assembly. Virion assembly occurs at the membrane in such a way that major coat protein subunits are packed in the filament as ␣-helices with their Nterminal regions on the outside and their C-terminal regions buried in the interior to interact with the DNA.One of the studies of the Pf1 virion by solid-state NMR was on magnetically aligned hydrated virions, and it characterized the path and orientation of the backbone of the ␣-helical major capsid protein subunit by means of 1 H-15 N polarization inversion spin exchange at the magic angle (PISEMA) (16,17). It was proposed that the subunit consists of an N terminus that forms a double hook, a C terminus with an unraveled ␣-helix, and a central portion of three ␣-helices with two bends near the center. An assumed capsid helical symmetry for the high-temperature form of the virion (6, 20) was used to generate the intersubunit contacts for a model of the capsid (16,17). In a more recent solid-state NMR study from our group (18, 19), m...

Correlation between viral production and carbon mineralization under nitrate-reducing conditions in aquifer sediment

“…These VLPs are morphologically similar to other filamentous bacteriophage. Similarly sized filamentous phage have been identified in pure culture as well as environmental samples: KSF-1F 1200 nm long 7 nm wide (Faruque et al, 2005), Pf1 2000 nm wide (Lorieau et al, 2008), VFJF 1400 nm long 7 nm wide (Wang et al 2013), B5 620 nm long 12 nm wide (Chopin et al, 2002); alpine lake filamentous viruses 450-730 nm long 30-40 nm wide (Hofer and Sommaruga, 2001). Together, these results indicate that Pseudomonas sp.…”

Helium-cooling and -spinning dynamic nuclear polarization for sensitivity-enhanced solid-state NMR at 14T and 30K

“…Uniquely, SSNMR does not require the sample systems to tumble fast isotropically as in liquid sample, or to preserve a static and long-range molecular alignment as in a single crystal, thus is applicable to samples not accessible with liquid-state NMR and X-ray crystallography. In particular, recent hardware and methodological advancements have enabled detailed SSNMR studies on biological supra-molecular assemblies such as proteins in lipid membrane [1][2][3][4], fibrous protein aggregates [5][6][7][8], and even intact viruses and organelles [9][10][11]. However, its low intrinsic sensitivity still presents a major bottleneck to many SSNMR studies.…”

Protein oligomers studied by solid‐state NMR – the case of the full‐length nucleoid‐associated protein histone‐like nucleoid structuring protein

“…So far, there have been only a few studies on protein systems known to interact with DNA or RNA. Quadrupolar ssNMR was used on a minimal DNA-binding domain of the human nucleotide excision repair protein XPA [21], and ssNMR was used to probe the protein structure and dynamics of Pf1 bacteriophage [22]. In the following, we show that high-resolution ssNMR spectra can be obtained from full length H-NS and partially assigned, providing atomic resolution probes located in different important regions of the protein sequence.…”