Structure similarity, difference and variability in the filamentous viruses fd, If1, IKe, Pf1 and Xf. Investigation by laser Raman spectroscopy

The filamentous bacteriophages fd, If1, IKe, Pf1, Xf and Pf3 in aqueous solutions of low, moderate and high ionic strength have been investigated as a function of temperature by laser Raman difference spectroscopy. By analogy with Raman spectra of model compounds and viruses of known structure, the data reveal the following structural features: the predominant secondary structure of the coat protein subunit in each virus is the alpha-helix, but the amount of alpha-helix differs from one virus to another, ranging from an estimated high of 100% in Pf1 to a low of approximately 50% in Xf. The molecular environment and intermolecular interactions of tyrosine, tryptophan and phenylalanine residues differ among the different viruses, as do the conformations of aliphatic amino acid side-chains. The foregoing features of coat protein structure are highly sensitive to changes in Na+ concentration, temperature or both. The backbones of A-DNA and B-DNA structures do not occur in any of the viruses, and unusual DNA structures are indicated for all six viruses. The alpha-helical protein subunits of Pf1, like those of Pf3 and Xf, can undergo reversible transitions to beta-sheet structures while retaining their association with DNA; yet fd, IKe and If1 do not undergo such transitions. Raman intensity changes with ionic strength or temperature suggest that transgauche rotations of aliphatic amino acid side-chains and stacking of aromatic side-chains are important structural variables in each virus.     

Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd

Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Conversely, the 229-nm UVRR spectrum is dominated by tyrosines (Tyr 25 and Tyr 40) of the 46-residue alpha-helical coat subunit. UVRR spectra excited at 244 and 238 nm exhibit Raman bands diagnostic of both viral DNA and coat protein tyrosines. Raman markers of packaged Pf1 DNA contrast sharply with those of the DNA packaged in the class I filamentous virus fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J., Jr. (1997) Biochemistry 36, 7810-7820]. Interestingly, deoxynucleotides of Pf1 DNA exhibit sugars in the C2'-endo/anti conformation and bases that are largely unstacked, compared with C3'-endo/anti conformers and very strong base stacking in fd DNA; hydrogen-bonding interactions of thymine carbonyls are also different in Pf1 and fd. On the other hand, coat protein tyrosines of Pf1 exhibit Raman markers of ring environment identical to those of fd, including an anomalous singlet at 853 cm-1 in lieu of the canonical Fermi doublet (850/830 cm-1) found in globular proteins. The results indicate markedly different modes of organization of ssDNA in Pf1 and fd virions, despite similar environments for coat protein tyrosines, and suggest strong hydrogen-bonding interactions between DNA bases and coat subunits of Pf1 but not between those of fd. We propose that structural relationships between the protein coat and encapsidated ssDNA genome are also fundamentally different in the two assemblies.     

Silver and mercury probing of deoxyribonucleic acid structures in the filamentous viruses fd, If1, IKe, Xf, Pf1, and Pf3

Ag+ binding and Hg2+ binding to both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) have been examined in some detail, and the results have been applied to study the structures of circular ssDNA in several filamentous viruses. It has been known for some time that Ag+ and Hg2+ bind to the bases of DNA producing characteristic large changes in absorbance and circular dichroism (CD) spectra, as well as changes in sedimentation rates. In the case of Ag+, it is known that there are three modes of binding to isolated dsDNA, referred to as types I, II, and III. Type III binding, by definition, occurs when Ag+ binds to Ag-dsDNA complexes having sites for binding types I and II extensively occupied, if not saturated. It produces CD spectra, assigned in this study, and absorbance spectra that are isosbestic with those of the Ag-dsDNA complexes present prior to its onset. In phosphate buffers binding is restricted to types I and II, whereas in borate buffers weaker type III binding can occur. Characteristics of types I, II, and III were observed for the DNAs in fd, If1, IKe, and Xf, but not for those in Pf1 and Pf3. Similarly, many of the spectral changes seen when Hg2+ binds to isolated double-stranded DNA are mimicked by Hg2+ binding to the DNAs within fd, IKe, If1, and Xf, but not for those in Pf1 and Pf3. The Ag+ and Hg2+ results indicate the presence of right-handed DNA helices in fd, If1, IKe, and Xf, with the two antiparallel strands of the covalently closed single-stranded DNAs having the bases directed toward the virion axes. For Pf1 and Pf3, Ag+ and Hg2+ binding cause large absorbance changes but only small CD changes. The very different results for Pf1 and Pf3 are consistent with the presence of inverted DNA structures (I-DNA) with the bases directed away from the structure axes, but the two structures differ from one another. Sedimentation velocity changes with Ag+ and Hg2+ binding strongly suggest structural linkages between the DNA and the surrounding protein sheath in each of the viruses.     

Structure and organization of bacteriophage Pf3 probed by Raman and ultraviolet resonance Raman spectroscopy

The Pseudomonas bacteriophage Pf3 is a long and narrow filament consisting of a covalently closed DNA single strand of 5833 bases sheathed by approximately 2500 copies of a 44-residue subunit. Ultraviolet resonance Raman spectra excited at 257, 244, 238, and 229 nm and off-resonance Raman spectra excited at 514.5 nm are reported for Pf3 in both H2O and D2O solutions. The key Raman bands are assigned to specific protein and DNA groups of the native virion assembly. The results are compared with proposed assembly models and Raman spectra recently reported for the isomorphous (class II) Pseudomonas phage Pf1 and the morphologically distinct (class I) coliphage fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J. , Jr. (1997) Biochemistry 36, 7810-7820; Wen, Z. Q., Armstrong, A., and Thomas, G. J., Jr. (1999) Biochemistry 38, 3148-3156]. Surprisingly, deoxynucleosides of the packaged DNA genome of Pf3 adopt the same conformation (C3'-endo/anti) found for DNA packaged in the class I fd virion rather than that (C2'-endo/anti) associated with DNA in the isomorphous Pf1 virion. However, DNA base stacking in Pf3, as judged by Raman hypochromic effects, differs significantly from that occurring in either Pf1 or fd. Thus, the single-stranded DNA genomes of Pf3, Pf1, and fd are all organized differently within their respective capsids, implying that local subunit-DNA interactions may be important in determining the structure specific to each native assembly. The present study confirms a completely alpha-helical secondary structure for the Pf3 subunit and an unusual indolyl ring environment for the subunit tryptophan residue (Trp-38).     

DNA packing in the filamentous viruses fd, Xf, Pf1 and Pf3.

Spectral data for filamentous viruses in the presence and absence of Ag+, together with other parameters, indicate that the DNA structures in two of the viruses, fd and Xf, are similar to each other but that these differ from two quite unusual and different DNA structures in Pf1 and Pf3.     

Quantitative analysis of nucleic acids, proteins, and viruses by Raman band deconvolution

A constrained, iterative Fourier deconvolution method is employed to enhance the resolution of Raman spectra of biological molecules for quantitative assessment of macromolecular secondary structures and hydrogen isotope exchange kinetics. In an application to the Pf1 filamentous bacterial virus, it is shown that the Raman amide I band contains no component other than that due to alpha-helix, indicating the virtual 100% helicity of coat proteins in the native virion. Comparative analysis of the amide I band of six filamentous phages (fd, If1, IKe, Pf1, Xf, and Pf3), all at the same experimental conditions, indicates that the subunit helix-percentage ranges from a high of 100% in Pf1 to a low of 71% in Xf. Deconvolution of amide I of Pf3 at elevated temperatures, for which an alpha-to-beta transition was previously reported (Thomas, G. J., Jr., and L. A. Day, 1981, Proc. Natl. Acad. Sci. USA., 78:2962-2966), allows quantitative evaluation of the contributions of both alpha-helix and beta-strand conformations to the structure of the thermally perturbed viral coat protein. Weak Raman lines of viral DNA bases and coat protein side chains, which are poorly resolved instrumentally, are also distinguished for all viruses by the deconvolution procedure. Application to the carbon-8 hydrogen isotope exchange reaction of a purine constituent of transfer RNA permits accurate determination of the exchange rate constant, which is in agreement with calculations based upon curve-fitting methods.     

Filamentous bacterial viruses. XI. Molecular architecture of the class II (Pf1, Xf) virion

The diffraction patterns of the Pf 1 and Xf strains of filamentous bacterial viruses (class II) can be interpreted in terms of a simple helix of protein subunits with 15Åpitch, having 22 units in five turns. The protein subunits are each elongated in an axial direction, and also slope radially, so as to overlap each other, giving an arrangement of subunits reminiscent of scales on a fish. The protein helix forms a tube with inner diameter about 20Åand outer diameter about 60Å. The single-stranded circular DNA is contained within this tube, with two DNA strands running the length of the tube.The diffraction patterns of fd, If 1 and IKe (class I) can be interpreted in terms of a perturbed version of the class II simple helix.     

Raman spectroscopy of filamentous bacteriophage Ff (fd, M13, f1) incorporating specifically-deuterated alanine and tryptophan side chains. Assignments and structural interpretation.

Structural interpretation of the Raman spectra of filamentous bacteriophages is dependent upon reliable assignments for the numerous Raman vibrational bands contributed from coat protein and packaged DNA of the virion. To establish unambiguous assignments and facilitate structural conclusions derived from them, we have initiated a systematic study of filamentous bacteriophage Ff (fd, f1, M13) incorporating protein subunits with specifically deuterated amino-acid side chains. Here, we report and interpret the Raman spectra of fd virions which incorporate: (a) a single deuterio-tryptophan residue per coat protomer [fd(Wd5)], (b) ten deuterio-alanines per protomer [fd(10Ad3)], and (c) both deuterio-tryptophan and deuterio-alanine [fd(Wd5 + 10Ad3)]. The unambiguous assignment of coat protein Raman bands in normal and deuterated isotopomers of fd establishes the validity of earlier empirical assignments of many key Raman markers, including those of packaged ssDNA (Thomas et al., 1988). Present results confirm that deoxyguanosine residues of the packaged ssDNA molecule depart from the usual C2'-endo/anti conformation characteristic of protein-free DNA in aqueous solution, although C2'-endo/anti conformers of thymidine are not excluded by the data. The combined results obtained here on normal fd, and on fd incorporating deuterio-tryptophan [fd(Wd5) and fd(Wd5 + 10Ad3)], show also that the microenvironment of the single tryptophan residue per coat protomer (W26) can be clearly deduced as follows: (a) The indole 1-NH donor group of each protomer in fd forms a moderately strong hydrogen bond, most likely to a hydroxyl oxygen acceptor. (b) The planar indole ring exists in a hydrophilic environment. (c) The torsion angle describing the orientation of the indole ring (C3-C2 linkage) with respect to the side-chain (C alpha-C beta bond) is unusually large, i.e., magnitude of X2,1 approximately 120 degrees. With respect to alanine isotopomers, the present results show that alanine residues, and possibly other methyl-containing side chains, are significant contributors to the fd Raman spectrum. The present study provides new information on protomer side chains of fd and demonstrates a Raman methodology which should be generally useful for investigating single-site interactions and macromolecular conformations in other nucleoprotein assemblies.     

Subunit secondary structure in filamentous viruses: predictions and observations

The algorithm of Garnier, Osguthorpe and Robson (J. Mol. Biol. 120, 97-120, 1978) for prediction of protein secondary structure has been applied to the coat protein sequences of six filamentous bacteriophages: fd, If1, IKe, Pf1, Xf and Pf3. For subunits of Class I virions (fd, If1, IKe), the algorithm predicts a very high percentage of helix in comparison to other structure types, which is in accord with the results of laser Raman and circular dichroism measurements. For subunits of the Class II virions (Pf1, Xf, Pf3), the algorithm consistently predicts a predominance of beta structure, which is compatible with the demonstrated facility for conversion of Class II subunits from alpha-helix to beta-strand under appropriate experimental conditions (Thomas, Prescott and Day, J. Mol. Biol. 165, 321-356, 1983). Even when the algorithm is biased to favor helix, the Class II virion subunits are predicted to contain considerably more strand than helix. Qualitatively similar results are obtained using the algorithm of Chou and Fasman (Adv. Enzym. 47, 45-148, 45-148). Therefore, both predictive and experimental methods indicate a distinction between Class I and II subunits, which is reflected in a greater tendency of the latter to adopt other than uniform alpha-helical conformation. The results suggest a possible model for the disassembly of filamentous viruses which may involve the unraveling of coat protein helices at the N terminus.     

Comparison of protein and deoxyribonucleic acid backbone structures in fd and Pf1 bacteriophages

The conformations of the protein and nucleic acid backbones in the filamentous viruses fd and Pf1 are characterized by one- and two-dimensional solid-state NMR experiments on oriented virus solutions. Striking differences are observed between fd and Pf1 in both their protein and DNA structures. The coat proteins of fd and Pf1 are almost entirely alpha helical and in both viruses most of the helix is oriented parallel to the filament axis. fd coat protein is one stretch of alpha helix that is slightly slued about the filament axis. In Pf1 coat protein two distinct sections of alpha helix are present, the smaller of which is tilted with respect to the filament axis by about 20 degrees. The DNA backbone structure of fd is completely disordered. By contrast, the DNA backbone of Pf1 is uniformly oriented such that all of the phosphodiester groups have the O-P-O plane of the nonesterified oxygens approximately perpendicular to the filament axis.     

Subunit Secondary Structure in Filamentous Viruses: Predictions and Observations

Abstract

The algorithm of Gamier, Osguthorpe and Robson (J. Mol. Biol. 120, 97–120, 1978) for prediction of protein secondary structure has been applied to the coat protein sequences of six filamentous bacteriophages: fd, Ifl, IKe, Pfl, Xf and Pf3. For subunits of Class I virions (fd, Ifl, IKe), the algorithm predicts a very high percentage of helix in comparison to other structure types, which is in accord with the results of laser Raman and circular dichroism measurements. For subunits of the Class II virions (Pfl, Xf, Pf3), the algorithm consistently predicts a predominance of β structure, which is compatible with the demonstrated facility for conversion of Class II subunits from α-helix to β-strand under appropriate experimental conditions (Thomas, Prescott and Day, J. Mol. Biol. 165, 321–356, 1983). Even when the algorithm is biased to favor helix, the Class II virion subunits are predicted to contain considerably more strand than helix. Qualitatively similar results are obtained using the algorithm of Chou and Fasman {Adv. Enzym. 47, 45–148,45-148). Therefore, both predictive and experimental methods indicate a distinction between Gass I and II subunits, which is reflected in a greater tendency of the latter to adopt other than uniform β-helical conformation. The results suggest a possible model for the disassembly of filamentous viruses which may involve the unraveling of coat protein helices at the N terminus.     

Different packaging of DNA in the filamentous viruses Pf1 and Xf

Xf Virus DNA, like Pf1 DNA, is a single-stranded circular molecule and contains, within experimental error, the same number of nucleotides, 7400. This was unexpected since Pf1 virus is 2 μm long while Xf virus is only 1 μm long. The ratio of nucleotides to major coat protein subunits has been found to be nearly unity in Pf1 and nearly two in Xf, but it is not certain that the ratios have exactly integer values. Calculations give the average axial internucleotide separation in Pf1virus as 5.3 Å whereas in Xf virus, the calculated separation is only 2.6 Å. The protein subunits in both Pf1 and Xf have calculated axial separations close to 2.6 Å. The results provide a solution to a problem encountered in the interpretation of X-ray diffraction patterns of these viruses concerning the number of protein subunits per helical turn.     

In vitro deletion mapping of the viral strand replication origin of Pseudomonas bacteriophage Pf3.

The origin of viral strand replication of the filamentous bacteriophage Pf3 has been characterized in Escherichia coli by in vitro deletion mapping techniques. The origin region was functionally identified by its ability to convey replicative properties to a recombinant plasmid in a polA host in which the replication origin of the vector plasmid is not functional. The origin of Pf3 viral strand replication is contained within a DNA sequence of 139 bp. This sequence covers almost completely one of the intergenic regions of the Pf3 genome, and it specifies both replication initiation and termination functions. Although no nucleotide sequence homology is present between the Pf3 origin of viral strand replication and that of the E. coli filamentous phages Ff (M13, f1, and fd) and IKe, their map positions and functional properties are very similar.     

Effects of virion and salt concentrations on the Raman signatures of filamentous phages fd, Pf1, Pf3, and PH75

Filamentous phages consist of a single-stranded DNA genome encapsidated by several thousand copies of a small alpha-helical coat protein subunit plus several copies of four minor proteins at the filament ends. The filamentous phages are important as cloning vectors, vehicles for peptide display, and substrates for macromolecular alignment. Effective use of a filamentous phage in such applications requires an understanding of experimental factors that may influence the propensity of viral filaments to laterally aggregate in solution. Because the Raman spectrum of a filamentous phage is strongly dependent on the relative orientation of the virion with respect to the polarization direction of the electromagnetic radiation employed to excite the spectrum, we have applied Raman spectroscopy to investigate lateral aggregation of phages fd, Pf1, Pf3, and PH75 in solution. The results show that lateral aggregation of the virions and anisotropic orientation of the aggregates are both disfavored by high concentrations of salt (>200 mM NaCl) in solutions containing a relatively low virion concentration (<10 mg/mL). Conversely, the formation of lateral aggregates and their anisotropic orientation are strongly favored by a low salt concentration (<0.1 mM NaCl), irrespective of the virion concentration over a wide range. The use of Raman polarization effects to distinguish isotropic and anisotropic solutions of filamentous phages is consistent with previously reported Raman analyses of virion structures in both solutions and fibers. The Raman data are supported by electron micrographs of negatively stained specimens of phage fd, which permit an independent assessment of salt effects on lateral aggregation. The present results also identify new Raman bands that serve as potential markers of subunit side-chain orientations in filamentous virus assemblies.     

Flow linear dichroism spectra of four filamentous bacteriophages: DNA and coat protein contributions

In this study, we have separated the contributions of DNA and protein to the absorption and linear dichroism (LD) of each of four phages: fd, IKe, Pf1, and Pf3. We have found that the DNA packaged in each of the phages is hypochromic relative to the purified single stranded DNA, suggesting that bases are stacked in all of the phages. We have oriented the phages by flow and for the first time report the intrinsic LD from 320 to 190 nm for each of these phages. From the intrinsic LD of the phages and the isotropic absorption of the individual components, we have determined the reduced dichroism of the DNA within the phages and, subsequently, the maximum angle of inclination of the DNA bases (from the helix axis) for the packaged DNA. The maximum angles were 63° and 64° for the DNAs of class I phages fd and IKe, respectively. The angles were significantly less, 51° and 49°, for the DNAs of the class II phages Pf1 and Pf3, respectively. Thus, the two classes of phage differ in the structures of their packaged DNA, the DNA bases of the class II phages being more parallel to the long axis of the phage than are the DNA bases of the class I phages.     

Structural characterization of the filamentous bacteriophage PH75 from Thermus thermophilus by Raman and UV-resonance Raman spectroscopy

The filamentous bacteriophage PH75, which infects the thermophile T. thermophilus, assembles in vivo at 70 degrees C and is stable to at least 90 degrees C. Although a high-resolution structure of PH75 is not available, the virion is known to comprise a closed single-stranded (ss) DNA circle of 6500 nucleotides sheathed by a capsid comprising 2700 copies of a 46-residue subunit (pVIII). Here, we employ Raman and UV-resonance Raman (UVRR) spectroscopy to identify structural details of the pVIII and DNA constituents of PH75 that may be related to the high thermostability of the native virion assembly. Analysis of the Raman amide I and amide III signatures reveals that the capsid subunit secondary structure is predominantly (87%) alpha-helical but contains a significant number of residues (6 +/- 1 or 13 +/- 3%) differing from the canonical alpha-helix. This minor structural component is not apparent in capsid subunits of the mesophilic filamentous phages, fd, Pf1, and Pf3, previously examined at similar spectral resolution. The Raman signature of PH75 also differs from those of fd, Pf1, and Pf3 by virtue of an unusual alanine marker (898 cm(-)(1) band), which is attributed to C(alpha)-H hydrogen-bond donation by subunit Ala residues. Because alanines of the PH75 subunit occur primarily within sXXXs motifs (where s is a small side chain, e.g. Gly, Ala, Ser), and because the occurrence of such motifs in alpha-helices is believed to thermostabilize interhelix associations via C(alpha)-H...O interactions [G. Kleiger et al. (2002) Biochemistry 41, 5990-5997], we propose that such hydrogen bonding may explain both the alanyl and amide I/III markers of PH75 capsid subunits and that C(alpha)-H...O interactions may serve as a significant source of virion thermostabilization. Raman and UVRR signatures of PH75 are also distinguished from those of fd, Pf1, and Pf3 by several marker bands that are indicative of hydrophilic Trp and Tyr environments, including hydrogen bonding interactions of aromatic ring substituents. These interactions are likewise proposed as contributors to the high thermostability of PH75 vis-a-vis fd, Pf1, and Pf3. Finally, PH75 is the only filamentous phage exhibiting UVRR markers diagnostic of a highly base-stacked ssDNA genome incorporating the low energy C2'-endo/anti deoxynucleoside conformation. The present results suggest that both intersubunit interactions and genome organization contribute to the enhanced thermostability of PH75 relative to mesophilic filamentous bacteriophages.     

Intensity of the polarized Raman band at 1340-1345 cm-1 as an indicator of protein alpha-helix orientation: application to Pf1 filamentous virus

Raman spectra of oriented alpha-helical protein molecules exhibit a prominent band near 1340-1345 cm(-)(1), the intensity of which is highly sensitive to molecular orientation. Polarization of the 1340-1345 cm(-)(1) marker is evident in Raman spectra of alpha-helical poly-L-alanine (alphaPLA) and alpha-helical poly-gamma-benzyl-L-glutamate (alphaPBLG). Corresponding polarization is also observed in Raman spectra of the filamentous virus Pf1, which is an assembly of alpha-helical coat protein molecules. In alphaPLA and alphaPBLG, we assign the band to a normal mode of symmetry type E(2) and specifically to a vibration localized in the (O=C)-C(alpha)-H linkages of the main chain peptide group. Although strict helical symmetry does not apply to coat subunits of filamentous viruses, an approximate E(2)-type mode may be presumed to account for a corresponding Raman band of Pf1 and fd filamentous viruses. Spectroscopic studies of N-methylacetamide and isotopically-edited fd viruses support the present assignment of the 1340-1345 cm(-)(1) band. Polarization anisotropy indicates that this band may be exploited as a novel indicator of protein alpha-helix orientation. Application of this approach to the polarized Raman spectrum of Pf1 suggests that, on average, the axis of the alpha-helical coat protein subunit in the native virion structure forms an angle of 20 +/- 10 degrees with respect to the virion axis.     

Crystal and solution structures of the B-DNA dodecamer d(CGCAAATTTGCG) probed by Raman spectroscopy: heterogeneity in the crystal structure does not persist in the solution structure

The self-complementary dodecamer d(CGCAAATTTGCG) crystallizes as a double helix of the B form and manifests a Raman spectrum with features not observed in Raman spectra of either DNA solutions or wet DNA fibers. A number of Raman bands are assigned to specific nucleoside sugar and phosphodiester conformations associated with this model B-DNA crystal structure. The Raman bands proposed as markers of the crystalline B-DNA structure are compared and contrasted with previously proposed markers of Z-DNA and A-DNA crystals. The results indicate that the three canonical forms of DNA can be readily distinguished by Raman spectroscopy. However, unlike Z-DNA and A-DNA, which retain their characteristic Raman fingerprints in aqueous solution, the B-DNA Raman spectrum is not completely conserved between crystal and solution states. The Raman spectra reveal greater heterogeneity of nucleoside conformations (sugar puckers) in the DNA molecules of the crystal structure than in those of the solution structure. The results are consistent with conversion of one-third of the dG residues from the C2'-endo/anti conformation in the solution structure to another conformation, deduced to be C1'-exo/anti, in the crystal. The dodecamer crystal also exhibits unusually broad Raman bands at 790 and 820 cm-1, associated with the geometry of the phosphodiester backbone and indicating a wider range of (alpha, zeta) backbone torsion angles in the crystal than in the solution structure. The results suggest that backbone torsion angles in the CGC and GCG sequences, which flank the central AAATTT sequence, are significantly different for crystal and solution structures, the former containing the greater diversity.(ABSTRACT TRUNCATED AT 250 WORDS)