Journal of Magnetic Resonance 253 (2015) 80–90

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Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface Gili Abramov, Omry Morag, Amir Goldbourt ⇑ School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel

a r t i c l e

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Article history: Received 26 October 2014 Revised 5 January 2015

Keywords: Bacteriophage viruses Magic-angle spinning Solid-state NMR Protein–DNA interactions Molecular assemblies Filamentous bacteriophage Phage

a b s t r a c t Bacteriophages are viruses that infect bacteria. They are complex macromolecular assemblies, which are composed of multiple protein subunits that protect genomic material and deliver it to specific hosts. Various biophysical techniques have been used to characterize their structure in order to unravel phage morphogenesis. Yet, most bacteriophages are non-crystalline and have very high molecular weights, in the order of tens of MegaDaltons. Therefore, complete atomic-resolution characterization on such systems that encompass both capsid and DNA is scarce. In this perspective article we demonstrate how magic-angle spinning solid-state NMR has and is used to characterize in detail bacteriophage viruses, including filamentous and icosahedral phage. We discuss the process of sample preparation, spectral assignment of both capsid and DNA and the use of chemical shifts and dipolar couplings to probe the capsid–DNA interface, describe capsid structure and dynamics and extract structural differences between viruses. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Bacteriophage viruses use bacterial cells to proliferate, thus they are widespread in all natural environments. The term bacteriophage derived from the words bacteria and phagein, which means ‘to eat’, most suitably describes the nature of virulent phages that cause cell lysis in the course of their life cycles. Some phages however are non-lytic and the cells remain intact after progeny release. Soon after their discovery nearly a century ago [1,2], it was realized that some phages can be used as therapeutic agents against bacterial infections [3]. Others are associated with various diseases [4,5]. Bacteriophages have played a role in some of the most significant discoveries and developments, amongst them the confirmation of DNA as the genetic material [6], the discovery of the genetic code for proteins [7], phage display [8–10] and recently bacteriophages were utilized for many bio- and nanotechnology applications [11–13]. Filamentous phage are also useful for the measurement of NMR anisotropic interactions in solution (e.g. residual dipolar couplings [14]) since they spontaneously align in magnetic fields. Many biochemical and biophysical properties of bacteriophages have been extensively reviewed [15–22], yet many of them remain structurally unresolved. Bacteriophage viruses share the general

⇑ Corresponding author. E-mail address: [email protected] (A. Goldbourt). http://dx.doi.org/10.1016/j.jmr.2015.01.011 1090-7807/Ó 2015 Elsevier Inc. All rights reserved.

construct of a central core consisting of a polynucleic acid chain surrounded by a protein coat, called the capsid. The capsid may be very simple in which case it is made of mostly a single coat protein arranged in a highly symmetric order, or quite complex, where it is composed of several types of symmetrically related subunits with different functions, which are arranged in a unique overall structure. The encapsulated genome can be a DNA or an RNA molecule either double- or single-stranded and varies significantly in size, from a few thousands of bases to hundreds of kilobase pairs. Individual components and whole structures of bacteriophages have been studied by a variety of techniques. Structural models are readily available for several icosahedral procapsids, empty capsids and rarely for full capsids from X-ray crystallography and cryo-EM. Some examples are the procapsid of the small (diameter of 25 nm) ssDNA icosahedral phage /X174 (pdb id 1CD3 [23]); the cryo-EM model of the empty capsid of the large (60 nm) dsDNA T7 icosahedral bacteriophage (pdb id 2XVR [24], see Fig. 1a); the crystal structure of the small RNA phage MS2 (2MS2 [25]), which also serves many times as a model to study protein– DNA interactions. There are numerous types of fibrous ssDNA filamentous phages [26], which are semi-flexible rods with lengths of approximately 1–2 lm (see Fig. 1b). The structures of a few filamentous viruses have been studied by X-ray fiber diffraction, cryo-EM and static solid-state NMR. They have been divided to two main structural classes [15–17] according to the fiber diffraction patterns; class-I phage have capsids that

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are made of many identical pentamers of the basic coat protein subunit related by an approximate twofold screw axis symmetry and are represented by the structures of Ff (fd, M13, f1) phage family (e.g. pdb id 2C0X [27]); in class-II phage protein subunits are arranged as monomers and have an overall helical capsid arrangement. The latter are represented by the structure of the Pf1 phage [28–30] (e.g. pdb ids 2XKM, 1PFI). Despite the sizable amount of structural information available on bacteriophage structure, many remain non-crystalline and unsolved, others only diffract to low resolution, some structures are non-conclusive and information on the DNA is lacking. It is well established that magic-angle spinning (MAS) solid-state NMR (ssNMR) provides high-quality data on structure and dynamics for high-molecular weight intact biocomplexes in native-like states [33–36]. Clearly, MAS ssNMR has advantages over other techniques to study bacteriophage systems, as it does not require crystallinity, alignment or cryogenic treatment. It therefore allows the study of viral particles under conditions closest to their natural environment. In this perspective, we discuss the application of MAS ssNMR to study several phage systems. We focus on two structurally different virus families; the filamentous ssDNA viruses Ff (fd, M13) and Pf1, and the dsDNA icosahedral phage T7. 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 [37–45] 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. 2. Capsid assignment and subunit conformation in filamentous phage Filamentous phage have molecular weights in the order of several tens of MDa and their capsids comprise over 80% of this mass. Their NMR spectra are therefore dominated by capsid resonances. Nonetheless, DNA signals are readily detected due to their unique

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chemical shifts (See Section 4). The capsid is essentially a multimer of thousands of symmetry-related copies of a 50 residues-long single coat protein subunit. Each signal of a specific atom is averaged over all protein subunits provided that subunits have a single uniform structure, or a small distinct number of conformations. The NMR results shown below support in large a single conformation, with the exception of some specific and localized chemical shifts indicating that the filamentous viruses discussed here (Pf1, fd, M13) essentially consist of a homogeneous capsid structure. 2.1. Sample preparation The bacteriophages discussed in this study can only assemble on their host bacteria and cannot be produced in vitro. Therefore, their production, labeling and extraction must follow the principles of common bacteriological protocols: bacterial growth, phage infection and phage purification. NMR experiments require the use of isotopically enriched samples. A common practice is to use 13C and 15N enrichment however, 2H-enriched media, not discussed here, provide a dilute proton environment and can be particularly useful for proton-detected ssNMR experiments [49]. The labeled samples are obtained by supplementing the minimal media of a bacterial culture with the relevant labeled precursors (e.g. 13C6glucose, 15NH4Cl). The phages then incorporate the specific labels into their capsids and genomes during their self-assembly process. Sparse labeling using partially labeled precursors (e.g. glycerol or glucose) can also be significant for completing the assignments and for obtaining long-range, inter-subunit contacts [50–52]. In particular, the use of (1,3-13C)-glycerol and (2-13C)-glycerol as labeling precursors of the M13 virus has led to an atomic-detailed quaternary structure of the phage capsid [32]. It is interesting to note in that respect that the labeling patterns of Pf1, the host of which is Pseudomonas aeruginosa, is different and results from the Entner–Doudoroff metabolic pathway [53,54]. Such a labeling pattern can also be induced in Escherichia coli facilitating similar enrichment patterns in overexpressed proteins [55]. After the growth of phage-infected bacteria, the labeled culture is centrifuged and the viruses in the solution are precipitated and subjected to cesium chloride gradient ultracentrifugation. The typical yield of purified phages (protein and nucleic acid) is 50–80 mg for Ff phages and 0.5 mg for T7 phage per liter of culture. 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]. 2.2. Chemical shift assignment

Fig. 1. (a) Top: the cryo-EM model of mature empty T7 capsid, which is composed of 415 copies of the coat protein subunits, arranged as pentamers on the vertices and hexamers on the faces of the icosahedron. Reproduced from the protein data bank [31], PDB id 2XVR [24]. Bottom: TEM image of wild-type fully packed T7 measured in Tel Aviv University. (b) Filamentous phage. Top: 35 subunits of M13 capsid [32] (PDB id 2MJZ). Bottom: TEM image of the M13 phage.

The chemical shifts of biological macromolecules provide information on structure, dynamics, hydrogen bonding, pKa etc. The interpretation can be based on the peak values, intensities and lineshapes of one or several experiments and samples. Site-specific resonance assignment is therefore key to understanding phage properties. Many pulse sequences aimed at chemical shift assignments have been developed and are based on the acquisition of multi-dimensional experiments involving well established dipolar-based and J-based correlations of 1H, 15N and 13C nuclei [33,56–59]. 13 C and 15N chemical shifts for bacteriophage capsids were obtained from 3D NCACX and NCOCX experiments and from 2D homonuclear 13C–13C RAD [60] (or DARR [61]), RFDR [62] and INADEQUATE [63] experiments acquired on uniformly 13C, 15N-enriched samples. The resulting spectra provided sequential contacts

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as well as sidechain assignment. The first assignment of a coat protein of an intact filamentous phage by MAS NMR was conducted on Pf1 [37] (BMRB accession number 15138), a class-II phage of unique structure due to the integer ratio of nucleotides to protein subunits. In two independent studies, the chemical shifts of the two closely related Ff virions fd [40] (BMRB 17728) and M13 [41] (BMRB 19747) have been obtained. Phages fd and M13 differ from Pf1 significantly by sequence and structure however, their own coat protein sequences differ only in a single amino acid – the negatively charged aspartate-12 in fd is replaced with a neutral asparagine in M13. Despite minor variations of their chemical shifts (see Section 3), the reduction of a single charge from every subunit has profound impact on their macroscopic behavior [64]. A characteristic 13C–13C DARR spectrum of the wild-type fd phage is given in Fig. 2a. As seen in this spectrum and as was observed also for Pf1 and M13, mostly narrow lines are obtained and single peaks for the vast majority of the assigned atoms in the coat protein are detected, suggesting a single conformation of the individual viral coat proteins and within different virus particles. Nevertheless, for very few sidechain atoms of specific residues in fd/M13 and in Pf1, chemical shift dispersion or chemical shift multiplicity was observed that suggests some localized polymorphism. In particular, those were observed in regions that are in contact with the DNA, such as Y40 and M42 in Pf1 [37,45] and Ile32 in M13 and fd [40,41]. These changes might suggest an effect of the different nucleotides on the chemical environment of those residues due to the tight packing of filamentous phage. In Pf1 for example, Y40 was suggested to be stacked with DNA bases [28,29]. 2.3. The helicity of filamentous phage coat proteins Analysis of chemical shift values can be interpreted in terms of secondary structure elements. Qualitative predictions, obtained by comparison between experimental chemical shifts and random

coil values [65,66], provide information on propensity to form helix or b-strand motifs. Further prediction of capping motifs and turns can be obtain with the software MICS that relies on a database of known structures and chemical shifts [67]. Such large training sets of proteins can provide quantitative estimation of torsion angles with programs such as TALOS+ [68] and PREDITOR [69]. Application of these strategies to the chemical shifts of Pf1, fd and M13 phage coat proteins (shown in Fig. 2b and c) reveals a curved right-handed a-helical conformation spanning most of the subunit. Using MICS, the helix in Pf1 is predicted to be capped with an N-terminal type VIII turn while in fd and M13 it is a type-II b-turn. 3. Chemical shift perturbations Very small atomic-scale variations can be detected simply by the observation of chemical shift perturbations (CSP). Therefore, CSP are useful in mapping protein binding interactions, pH and temperature effects, small structural changes etc. This approach was applied to study the structural phase transition in Pf1 and to compare fd and M13 phages. Pf1 undergoes a reversible structural phase transition at 10 °C, in which the symmetry of the capsid changes [70–72]. Pf1 was prepared for MAS NMR studies above and below 10 °C and 13C homonuclear correlation experiments were performed at these two states [38]. Chemical shift perturbations were then mapped into the low temperature fiber diffraction models of Pf1 (pdb id 4IFM [73], 1PFI [29]) and compared to the lower resolution high temperature models (1QL1 [74], 2IFN [73]). CSP were observed in three patches that belong to the hydrophobic inter-subunit interface. Moreover, the residues that undergo the most significant changes also exhibit strong inter-subunit sidechains contacts and demonstrate how the structural phase transition is driven by structural changes of the main hydrophobic interaction interface within the capsid.

Fig. 2. Chemical shifts and secondary structure analysis of phage coat proteins. (a) 13C–13C DARR spectrum of wild-type fd virion showing chemical shifts of the coat protein in the aliphatic region. The 50-amino-acids sequence of the coat protein is given on top. A complete sidechain pattern of the single Pro6 residue is marked in the spectrum demonstrating the highly symmetric and homogeneous nature of the capsid. The broadened feature of I32 demonstrates that some heterogeneity exists in regions facing the DNA. The assignments of A27 and A10 have been revised in this work. (b) Secondary shifts for M13 phage. (c) Torsion angle prediction for the M13 capsid subunit using TALOS+. Torsion angle regimes for an ideal straight helix and a curved helix are indicated with black lines and gray rectangles, respectively. These results indicate a curved ahelical M13 (and fd) coat protein between residues 6–48.

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A comparison of fd and M13 was also performed by the CSP approach [41]. There are two notable differences between the samples used for MAS NMR studies: (i) a natural difference is the replacement in position 12 of charged aspartate in fd with a noncharged asparagine in M13; (ii) the phages had different total lengths (30%) due to DNA sequence modifications. The 13C–13C spectral overlay of fd and M13 is shown in Fig. 3. The two spectra exhibit extensive similarity despite their significantly different lengths. Most backbone and sidechain atoms, including the C-terminal lysine residues located at the DNA interface, overlap within 0.1 ppm. Such variations are small even for repetitive preparations of crystalline proteins suggesting a similar subunit packing in the hydrophobic regions along the capsid, and a similar capsid–DNA interface. Interestingly, magnetization buildup curves are similar as well (see Fig. 6 in Section 6). These results indicate that the phage assembly is well preserved and very stable and that the structures of M13 and fd are highly similar. Within the ‘‘bulk’’ of similar shifts, some notable perturbations were detected for the positively charged Lys8 sidechain and in vicinal residues, particularly Asx12 and Tyr24. We mapped the changes onto the available structural models of mutant fd (Y21M). CSP reveal that the elimination of the charge modifies electrostatic interactions and some hydrogen-bonding patterns. These results have further implications on capsid dynamics, as discussed in Section 6. As this small perturbation is amplified through the existence of thousands of subunits within the entire virion, it is likely to play a role not only in determining the exact symmetry of the capsid but also in the macroscopic liquid crystal structures formed by these phages. 4. Large encapsulated DNAs: System-driven solid-state NMR methodologies Genomes encapsulated in bacteriophage viruses vary significantly in size, conformation, and organization. Detailed structural information on viral genomes in native states is not easily accessible and the secondary structures of encapsulated DNAs were primarily studied by Raman spectroscopy, circular dichroism, and more recently by ssNMR [42,43,45]. Prior studies on tailed dsDNA phages show that the mechanisms of viral genome packaging and release are similar presumably since their genomes are highly condensed to a comparable degree [75]. Moreover, since the genomes are confined to capsids with comparable dimensions to their persistence lengths, they are most likely found in a twisted conformation. T7 is an example for a tailed dsDNA virus with such

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characteristics. Its 40 kbp dsDNA molecule comprises over half of the total viral mass, and it is coaxially spooled around an internal core within the capsid [76]. In many ways, the genome of T7 can be thought of as a paradigm structure for natural, highly compacted and bent DNA in functional biological assemblies. Unlike the classical head-and-tail phages the circular ssDNA in filamentous phage comprises only between 6% and 14% of the total viral mass and all nucleotides are in contact with the capsid subunits making protein–DNA interactions in such virions a key feature of their structure and of their assembly mechanism. Empty capsids have not been observed for filamentous viruses; DNA packing in the cell prior to assembly and out of the cell in the mature form are different and the DNA is capped by different capsids. Various biophysical studies on DNA structures in these viruses, particularly on fd and Pf1, reported on significantly different structural features for the DNA conformations presumably due to the different nucleotideto-subunit ratio of these two phages [77–79]. While capsid chemical shifts provide key information on phage structure, further characterization of the genomic material and of its interaction with the capsid is necessary for fully comprehending phage assembly and morphogenesis. Analogously to proteins, where amino acid shifts are used to determine secondary and even tertiary structures, the chemical shifts of DNA and RNA nucleotides are reporters of the deoxyribose puckering, the glycosidic bond orientation, base pairing and base stacking [80]. However, unlike the extensive database of amino acids shifts, nucleic acids database is scant, particularly for 13C and 15N atoms, and limited to small DNA/ RNA molecules, mostly in solution but also using ssNMR. In the following we describe ssNMR approaches toward the characterization of large genomes in the context of native bioassemblies. The methodologies are applied to the genomes of bacteriophages T7, fd and Pf1. 4.1. Labeling approaches for studying bacteriophage DNA In biomolecular complexes containing both proteins and nucleic acids, the DNA/RNA carbons and nitrogens resonate mostly at separated spectral regions from amino acids, yet some overlap exists (bases with aromatic carbons, ribose C20 and C50 with aliphatic carbons, 15N amine signals). In the T7 dsDNA phage the dominant DNA resonances could be clearly resolved from the weaker capsid peaks [44]. However, when the coat protein is large in mass compared to the genome, such as in the case of filamentous phage, blanking of amino acids is essential for obtaining unambiguous DNA shifts. Blanking, or unlabeling, is easily obtained when the bacterial mini-

Fig. 3. Similarities and differences within fd and M13 phages. Extracted regions from 2D 13C–13C DARR spectra of intact M13 (blue) and fd (red) bacteriophages. (a) Sequential contacts associated with glycine residues; (b) backbone and backbone-sidechain contacts; (c) hydrophobic sidechains. Lys8 cross-peaks are indicated in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Nucleotide-type assignments of DNA in large bacteriophage systems. (a) 15N–13C TEDOR spectrum showing mostly nucleotide correlations in wild-type fully labeled T7. The cryo-EM image of the spooled dsDNA in T7, showing its high homogeneity, was reprinted from [76]. (b) 13C–13C CORD correlation spectrum of aromatic-unlabeled fd virus showing the DNA region. (c) DNP-enhanced 13C–13C DARR spectrum of Pf1. Base and ribose correlation regions are shown on the left and right, respectively. (d) Nomenclature of the deoxyribose ring and the four types of nucleotides. The arrows show magnetization pathways of the spectrum in (a). The ribose is shown as a C20 -endo, as it appears in all phage discussed here.

mal media is supplemented with natural abundance amino acids in addition to 13C-glucose or any other labeled precursors. In the case of Pf1 [45], a minimal medium containing U–13C/15N glucose and NaH13CO3 was supplemented with twelve unlabeled amino acids, and resulted in a spectrum containing almost only ribose signals while base signals were missing probably since their biosynthesis requires amino acids. Aromatic-only unlabeling (with no apparent reduction in the signal of other residues) was used to study the capsid–DNA interface (see Section 5 and Fig. 5) of fd bacteriophage by supplementing the minimal medium with unlabeled tyrosine, phenylalanine and tryptophan [42]. 4.2. Chemical shifts in bacteriophage DNA Standard techniques for site-specific chemical shift assignment of proteins rely on the initial identification of spectrally isolated ‘anchor’ residues, or residue pairs, and consequently on backbone-walks allowing individual amino acids to be recognized according to their order in the protein sequence. Phage DNA consists of thousands of copies of four nucleotides. Therefore, it is not possible to obtain site-specific assignment and we focus on nucleotide-type assignments of the purines adenine (A) and guanine (G), of the pyrimidines cytosine (C) and thymine (T) and of the attached ribose moieties. Nonetheless, important information can be obtained, as detailed below, and identification of site multiplicity can be interpreted in terms of different DNA conformations,

or the existence of broad lines (with low dynamic order parameters) may indicate a non-uniform DNA organization. DNA 13C and 15N chemical shifts of bacteriophage samples were obtained from homonuclear 13C–13C spectra using sequences such as DARR or CORD [81] and from 15N–13C heteronuclear TEDOR [82]. While double-cross polarization 15N–13C experiments [83,84] are routinely used for protein assignment, they are less applicable for the assignment of nitrogen and carbon DNA atoms due to their selectivity toward narrow band widths (i.e. selecting N–CO or N– CA transfers in proteins). Some representative DNA spectra are shown in Fig. 4. Very much like in proteins, anchor base atoms for assignment can be found that have unique shifts (within more than ± 5 ppm on average). The thymine methyl carbon (TC7) and CC5 are completely isolated with no overlap with other nucleotide resonances and therefore these signals can be used for assignment of the pyrimidine nucleotides. The purines (G and A) can be identified unambiguously through cross-peaks of GC8 and AC8, which resonate on average 4 ppm apart. Since homonuclear experiments depend on proton-assisted magnetization transfer and many DNA base carbons are quaternary, many cross-peaks involving unprotonated atoms have very weak or missing autocorrelation peaks and become non-symmetric across the diagonal. Such properties can be used for the identification of the desired spin-systems. Alternatively, weak signal intensities can be alleviated by using sensitivity enhancement techniques including dynamic nuclear polarization (DNP), as was realized in the case of Pf1 phage.

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In Pf1, a combined ssNMR and DNP-NMR study at various fields and temperatures provided chemical shift assignments for most carbon and nitrogen resonances in the DNA [45]. A representative DNP-NMR spectrum showing the intra-base (with some base-sugar cross-peaks) and intra-ribose correlations demonstrate the considerable signal enhancement (22-fold) obtained by DNP alongside signal broadening, which was accompanied by clear splittings associated with heterogeneity of the DNA at low temperatures. For fd, a close to complete assignment of carbon resonances including nucleotide-specific ribose shifts was obtained (without DNP) at room temperature by performing CORD experiments on aromatic-unlabeled samples [42]. These shifts have been utilized to study the interface of the capsid with the DNA, as detailed in Section 5. Chemical shifts for the DNA in both fd and Pf1 have been analyzed in view of their basic conformational characteristics using current knowledge on the relationship between nucleic acid shifts and DNA structure [80]. The ribose chemical shifts are reliable reporters for the different sugar conformations; C30 and C50 are the most sensitive atoms shifting upfield by 7 and 5 ppm respectively in going from C20 -endo to C30 -endo conformations (S-type to N-type) [85]. Since quaternary carbons and tertiary amines have a small amount of reported shifts, interpretation of the results, in particular those of base atoms, must be taken with caution until the databases are sufficiently adequate. Although fd and Pf1 have very different DNA structures, according to NMR the two phages share a similar ribose sugar pucker (S-type) and an anti glycosidic bond conformation. Base shifts on the other hand, which are reporters of base stacking and hydrogen bonding, point to their existence in fd and less so in Pf1. Since chemical shifts are averaged over all bases according to their particular type, minor occupations of different conformations are possible but are below detection threshold of current data. Chemical shifts have also been obtained for the icosahedral bacteriophage T7 using a set of 2D experiments [44]. In T7, the DNA is double-stranded according to Raman data [86]. The heteronuclear 15 N–13C TEDOR spectrum of T7 (Fig. 4) reveals portions of the dsDNA assignment including the glycosidic tertiary amines. Such spectra demonstrate how through the 15N resonances, the ribose carbons C10 can be resolved from C40 , otherwise overlapping in the 13C spectra. Despite the inherent differences between the genomes of fd and T7 (circular ssDNA vs. spooled dsDNA), we find striking similarities between their secondary structure elements. Both share B-form DNA features, i.e. an S-type sugar pucker, an anti base orientation, and base pairing. Interestingly, sugar carbon shifts for the two phages are in the high end of B-form oligonucleotides shifts, in addition to their similar carbon base shifts. In summary, chemical shifts of DNA in large systems such as bacteriophages carry key information on secondary structure, on heterogeneity and potentially on dynamics. Some of the trends we observed in the systems described here are consistent with general knowledge while some cannot be easily explained, in particular the disagreement with Raman data (for fd) and the similarity of fd and T7. Further studies on similar complex protein–DNA assemblies, which will create a reliable chemical shift database for large native DNA molecules, as well as more computational studies [87], will accelerate efforts toward the characterization of complex DNA and RNA molecules in the context of intact biological assemblies.

5. Capsid–DNA interface Interactions between the capsid and the negatively charged genome can be directly observed by ssNMR using through-space

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polarization transfer techniques or by observing peak perturbations. In filamentous phage very close contacts exist between all nucleotides and the coat protein subunits while in icosahedral phage only the surface of the spooled DNA directly attaches to the capsid. It is therefore not surprising that empty capsids form prior to packaging in icosahedral viruses and can be isolated individually while in filamentous viruses the capsid subunits are attached to the DNA during assembly. In a ssNMR study of the icosahedral phage T4, Yu and Schaefer [43] used 15N{31P} and 31P{15N} REDOR [88] experiments on lyophilized 15N-lysine-labeled phage, and on unlabeled phage supplemented with 15NH+4; they observed correlations between positively charged amines and the negatively charged genomic phosphates. These experiments provided evidence for the partial charge balancing of the DNA in T4 and suggested electrostatics as a driving force for DNA packaging. Electrostatic interactions between capsid lysine sidechains and the DNA phosphate groups have also been detected in the filamentous phage fd. These contacts have been observed by two complementary techniques: 13C–13C CORD experiments, which were performed with long mixing times; 31P–13C correlations, which were detected using a PHHC experiment; here polarization originating from the DNA phosphates is transferred via protons to the capsid carbon resonances (the sequence is based on the CHHC experiment of Spiess and coworkers [89]). Both methods relied on the prior assignment of both capsid and DNA (Sections 3 and 4). Due to the tight DNA–capsid packing in filamentous phage, those experiments also report on the orientation of the capsid with respect to the viral axis since only residues facing the DNA produce observable signals in the spectrum, as shown in Fig. 5. Moreover, due to the special symmetry of fd, in which pentamers of identical subunits are related by an approximate twofold screw axis, analysis of existing cross-peaks provides crude information on the rise between pentamers. In the case of fd, cross-peaks with DNA were observed in the C-terminal part for the charged lysines, for the residues in the bottom end of the coat protein and for two hydrophobic amino acids facing the interior of the phage (Val33 and Ile37). No contacts with the DNA have been detected for residues preceding Val33 suggesting that the position of the next pentamer must lie no more than a few angstrom above this position. Indeed these results are in agreement with current phage models. Water plays a vital role in any protein assembly and the DNA hydration level determines its conformation. Solid-state NMR pulse schemes that correlate water protons with proteins are mainly based on HETCOR experiments combined with relaxation filters [90] that separate mobile from rigid water molecules. Such schemes allow to differentiate between water populations and to probe exchange processes [91,92]. For example, Bockmann and coworkers applied HETCOR and exchange spectroscopy to a PEG-crystallized Crh protein and were able to distinguish between unbound supernatant water and crystal-bound water pools [93,94]. It has been shown that the hydration level of filamentous bacteriophage viruses affects their symmetry [95] and that water inside the virion are likely in contact with distinct charged and protonated amino acids [39,96,97]. Preliminary work in our lab using 1H–13C MELODI–HETCOR [98] experiments applied to M13 phage also suggest similar effects (unpublished). An extensive experimental study of hydration water in Pf1 phage has recently been published [97]; Several HETCOR experiments, freezing experiments, chemical exchange and solvent removal experiments show that internal hydration water are in contact with many residues as well as with DNA. Hydration water observed at the C-terminus, attached to residues in close proximity to the DNA suggest that water plays a role in the capsid–DNA interface serving as a dielectric through

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Fig. 5. Capsid–DNA interactions. (a) 13C–13C CORD spectrum of aromatic-unlabeled fd showing the base-capsid correlations. (b) PHHC spectrum with marked correlation regions. Capsid–DNA cross-peaks are indicated in red. (c) 1H–13C MELODI–HETCOR spectrum of Pf1 showing water–capsid interactions. Residues involved in those interactions are labeled and colored on a single capsid monomer. Reprinted with permission from [97]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

which DNA and capsid charges interact. Fig. 5c shows the 1 H–13C MELODI–HETCOR spectrum of hydrated Pf1 and a single-subunit model revealing the hydrated amino acids. Water– capsid resonances involve N-terminal residues and a cluster of C-terminal residues that face inwards (toward the DNA) according to the Pf1 structural models. Such contacts, in conjugation with the detection of ribose correlations to the same proton signal, indicate the presence of hydration water in the protein– DNA interface. 6. Probing phage dynamics NMR is a very sensitive technique to measure motion. Through analysis of relaxation rates, exchange and averaged couplings of anisotropic interactions, dynamic motions spanning several orders of magnitude can be accessed [99,100]. Motions up to the submicrosecond timescale can be measured by comparing the dipolar coupling of a particular bond to the rigid limit value defined by common protein C–H, C–C, N–H or N–C distances. Therefore, measurements of the initial rise of single bonds in CC spin-diffusion spectra, of the H–X couplings by a technique such as separated local field spectroscopy and of NC couplings by REDOR or similar methods report on the dynamics of the backbone and sidechains of a protein. In bacteriophages, prior studies by static ssNMR of aligned fd samples suggested that the N-terminus is mobile [101] and the lack of electron density in fiber diffraction and cryo-EM studies further supports this evidence. Analysis of spin-diffusion CC buildup curves of both M13 and fd performed in our group [40,41] supports a mobile N-terminus as well since the signal intensity increases slowly and reaches lower maximal values (See Fig. 6a1). In Pf1 on the other hand, measurements of CH couplings were performed [102] by using Lee–Goldburg crosspolarization (LGCP) experiments [103], combined with 2D DARR spin diffusion. Results indicated that although the N-terminus is non-helical, as in fd, it is rigid on the submicrosecond time scale [39]. Furthermore, the entire backbone in Pf1 was shown to be entirely rigid as well as sidechains involved in hydrophobic

packing interactions. Mobile sidechains have been observed for solvent exposed residues and interestingly, for charged residues in contact with the DNA. In view of the capsid–DNA interactions discussed in the previous Section, this mobility is associated with the existence of water molecules mediating the electrostatic interactions with the DNA. In Section 3 we used chemical shift perturbations to compare fd and M13 in order to understand the effect of the D12N mutation on their structure. Very much in line with these results, the comparison of buildup curves for many isolated cross-peaks leads to the same conclusions; atoms having similar shifts exhibit similar dynamic properties, even for the mobile N-terminus and for sidechains pointing either inward to the DNA or participating in subunit packing. The fact that even sequential and long-range cross-peaks have similar intensities suggest that spin diffusion along increased distances is also unaffected hence the structures are similar not only structurally but also in terms of their internal motion. On the other hand, variations in chemical shifts are associated with variations in dynamics, in particular the sidechain of Lys8 (Fig. 5), which shows the most significant chemical shift deviation. The data suggest that in M13 the lysine sidechain is more rigid and presumably the elimination of the charge on the aspartate creates a denser network of hydrogen bonding and charges in M13, which involves the close N-terminal negatively charged residues as well as Glu20 and Tyr24 from different capsid subunits. 7. A case study: Silver-doped fd affects protein–DNA interactions We have shown how the chemical shift values and intensities of bacteriophage viruses carry a substantial amount of information. They are reporters of secondary structure, of protein–DNA interactions, of phage dynamics and of phage modifications. In this Section we demonstrate how perturbing a filamentous phage by silver ions affects the interactions of the capsid with the DNA, an observation that may reflect a macroscopic scale change since it was shown that this procedure increases and eventually unwinds the pitch of its spontaneously formed cholesteric phase [104].

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Fig. 6. Dynamics in filamentous phage capsid. (a) Buildup of CC polarization in DARR spectra comparing fd (red/filled symbols) and M13 (blue/open symbols) phage. (a1) Glycine residues including mobile G3; (a2) similar isoleucine sidechain dynamics; (a3) variations in lysine-8 sidechain dynamics. The cross-peak intensities for fd and M13 were normalized separately to their Gly23Ca-C0 shift. (b) Dipolar order parameters from LGCP curves indicate the rigidity of Pf1 capsid backbone. Part (b) is reproduced from Fig. 1 of [39]. Copyright Ó by the National Academy of Sciences. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

For this study we prepared two phage samples, a wild-type fd virus and a sample of fd that was doped with 1.73 mM of silver ions accounting to 1.2 silver ions per nucleotide (this value corresponds to a very large increase of the cholesteric pitch of fd at high concentrations and to a transition to nematic phase in a low concentration, 20 mg/mL [104]). The 13C–13C DARR correlation spectrum of the two samples was then measured at similar conditions and an overlay is shown in the top of Fig. 7. Two clear properties can be observed in the spectrum; chemical shift perturbations are almost not observed and can be pinned down to only lysine-8; however, peak intensities vary, at some positions significantly. Such changes are related to variations in spin dynamics and therefore we examined the locations of those changes. In Fig. 7b we show the ratio of peak intensities (on a logarithmic scale) of the two samples for many well-resolved cross-peaks that could be unambiguously assigned. In order to eliminate the effects of different sample amounts, we examined a region in which very little changes were observed and the average was used to normalize the data. Signals with significant (more than two standard deviations of the average) reduction of their intensity upon Ag+ titration are explicitly indicated and their sidechains are shown in Fig. 7c. Interestingly, all significant changes in the C-terminus occur for sidechains of residues in contact with the DNA. They suggest that upon complexation with silver the charge balance on the DNA is modified and therefore lysine sidechains are not tightly bound to the DNA phosphates. It is also interesting to note that even hydrophobic residues that point inwards according to our previous studies (Section 5) are also more mobile and some to an overall effect of loosening the capsid–DNA interface. Other changes can be observed in the N-terminus including the stronger signal of the negatively charged Glu2 and the weaker signals of Lys8 (the sidechains of

lys8 and Cd of E20 also undergo significant peak shifts) and to some extent Y24. These two residues are part of an electrostatic interaction network involving charged residues and hydrogen bonding. Similarly to the removal of the aspartate charge when changing fd to M13, here the addition of positive charges have a similar effect. Overall, most of the changes we observe upon silver binding are related to conformational flexibility and therefore we can conclude that while structural changes in the subunit are hardly detected, modification of the charge balance affects the DNA–capsid interface and the electrostatic interactions between subunits. Undoubtfully further ssNMR studies of DNA and capsid perturbants will provide insight into the relation of phage microscopic changes and its liquid crystal formation characteristics. 8. Summary Bacteriophage systems reviewed in this perspective show the applicability of MAS solid-state NMR studies to provide atomic-scale knowledge on intact viruses. The capsids, genomes, and protein–DNA interface of distinct bacteriophages were characterized by using existing and specifically designed pulse sequences and labeling schemes. Such data open routes for obtaining complete high-resolution three-dimensional structures of bacteriophage viruses and of other complex protein–DNA bioassemblies, which are commonly found in nature. Note added in proof: The NMR/Rosetta model of M13 bacteriophage Upon completion of the final revised form of this manuscript, the high-resolution structure of the M13 bacteriophage capsid

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Fig. 7. Ag+-doped fd phage. (a) Overlay of DARR spectra (blue – wild-type fd; red – Ag+-doped fd) acquired with a mixing time of 15 ms. (b) Logarithmic plot of peak intensity ratios. Normalization was obtained by averaging cross-peaks belonging to residues F11–V31, which show little variations. One standard deviation of peak intensities in this region is shaded in gray throughout the plot and a region corresponding to two standard deviations is indicated by the dashed lines. Cross-peaks with significant peak reduction upon silver doping are indicated. (c) A top view of the pentameric unit in the structural model of M13 phage (2MJZ) showing the capsid residues that are affected by Ag+ doping. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

has been published [32]. The structure was based on MAS NMR restraints (obtained from homonuclear 13C–13C spectra of sparsely labeled samples), on the C5 pentamer symmetry reported by fiber diffraction [105] and on the Rosetta fold-and-dock modeling protocol [106]. The structure reveals a quadrupoled binding epitope; each subunit participates in four identical hydrophobic/ aromatic pockets by contributing amino acids from different regions along the sequence. This binding epitope is not only key for the structure elucidation, but it may play a key role in phage morphogenesis and sustainability. Sequence homogloy suggests that such a packing is a common motif among various filamentous phage. The structure has been deposited in the protein data bank with id 2MJZ.

Acknowledgments We thank Dr. Loren Day for introducing us to filamentous viruses, for providing the various Pf1 samples and for teaching us the molecular biology of bacteriophages. Prof. Ann McDermott from Columbia University is acknowledged for all the work on Pf1. The fd phage construct fth1 was a kind gift from Prof. Jonathan Gershoni from the Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University. The M13 construct M13K07 and DH5aF0 host cells for both fd and M13 were a kind gift from Prof. Itai Benhar, Department of Microbiology and Biotechnology, Tel Aviv University. T7 and host was a kind gift from Dr. Udi Qimron from the Department of Human Microbiology, Faculty of Medicine, Tel Aviv University. The research on fd, M13 and T7 was supported by the Israel Science foundation grants

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