Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid.

P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses. The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure. Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups. Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed [Tuma, R., Prevelige, P. E., Jr., and Thomas, G. J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9885-9890]. Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations. Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering. Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies. We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone. We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice. However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core. The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit. The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid.

Orientations of Tyr 21 and Tyr 24 in the capsid of filamentous virus Ff determined by polarized Raman spectroscopy.

The capsid of filamentous virus Ff is assembled from approximately 2750 copies of a 50-residue alpha-helical subunit, the two tyrosines of which (Tyr 21 and Tyr 24) are located within a hydrophobic sequence that constitutes the subunit interface. We have determined the side chain orientations of Tyr 21 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, utilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman signature of Tyr 21 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signature of Tyr 24 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 2200-2400 cm(-1) interval of the Raman spectrum were measured and interpreted using tensors transferred from a polarized Raman analysis of L-tyrosine-2,3,5,6-d(4) single crystals. Polarized Raman analysis was extended to the bands of Ff near 642 and 855 cm(-1), which originate from vibrational modes of the tyrosine phenolic ring. The results indicate the following: (i) For both Tyr 21 and Tyr 24, the phenolic 2-fold axis (C(1)-C(4) line) is inclined at 41 +/- 5 degrees from the virion axis and the normal to the plane of the phenolic ring is inclined at 71 +/- 5 degrees from the virion axis; (ii) the mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman markers of the subunit tryptophan (Trp 26), suggesting interdependence of Tyr 24 and Trp 26 orientations in native Ff; and (iii) polarization anisotropies observed for Raman markers of Ff DNA bases are unperturbed by mutation of either Tyr 21 or Tyr 24, indicating that nonrandom base orientations of packaged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A molecular model consistent with these findings is proposed.

Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy.

Four genomic DNAs of differing GC content (Micrococcus luteus, 72% GC; Escherichia coli, 50% GC; calf thymus, 42% GC; Clostridium perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines spermidine ([H(3)N(CH(2))(3)NH(2)(CH(2))(4)NH(3)](3+)) and spermine ([(CH(2))(4)(NH(2)(CH(2))(3)NH(3))(2)](4+)). In solutions containing 60 mM DNA phosphate (approximately 20 mg DNA/ml) and either 1, 5 or 60 mM polyamine, only Raman bands associated with the phosphates exhibit large spectral changes, demonstrating that B-DNA phosphates are the primary targets of interaction. Phosphate perturbations, which are independent of base composition, are consistent with a model of non-specific cation binding in which delocalized polyamines diffuse along DNA while confined by the strong electrostatic potential gradient perpendicular to the helix axis. This finding provides experimental support for models in which polyamine-induced DNA condensation is driven by non-specific electrostatic binding. The Raman spectra also demonstrate that major groove sites (guanine N7 and thymine C5H(3)) are less affected than phosphates by polyamine-DNA interactions. Modest dependence of polyamine binding on genome base composition suggests that sequence context plays only a secondary role in recognition. Importantly, the results demonstrate that polyamine binding has a negligible effect on the native B-form secondary structure. The capability of spermidine or spermine to bind and condense genomic B-DNA without disrupting the native structure must be taken into account when considering DNA organization within bacterial nucleoids or cell nuclei.

Protein-directed DNA structure. I. Raman spectroscopy of a high-mobility-group box with application to human sex reversal.

Protein-directed reorganization of DNA underlies mechanisms of transcription, replication, and recombination. A molecular model for DNA reorganization in the regulation of gene expression is provided by the sequence-specific high-mobility-group (HMG) box. Structures of HMG-box complexes with DNA are characterized by expansion of the minor groove, sharp bending toward the major groove, and local unwinding of the double helix. The Raman vibrational signature of such DNA reorganization has been identified in a study of the SRY HMG box, encoded by the human male-determining region of the Y chromosome. We observe in the human SRY-HMG:DNA complex extraordinarily large perturbations to Raman bands associated with vibrational modes of the DNA backbone and accompanying large increases in intensities of Raman bands attributable to base unstacking. In contrast, DNA major-groove binding, as occurs for the bZIP protein GCN4 [Benevides, J. M., Li, T., Lu, X.-J., Srinivasan, A. R., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 548-556], perturbs the Raman signature of DNA only marginally. Raman markers of minor-groove recognition in the human SRY-HMG:DNA complex are due primarily to perturbation of specific vibrational modes of deoxyribose moieties and presumably reflect desolvation at the nonpolar interface of protein and DNA. These Raman markers may be diagnostic of protein-induced DNA bending and are proposed as a baseline for comparative analysis of mutations in SRY that cause human sex reversal.

Protein-directed DNA structure II. Raman spectroscopy of a leucine zipper bZIP complex.

Mechanisms of transcription may involve protein-directed changes in DNA structure and DNA-directed changes in protein structure. We have employed Raman spectroscopy to characterize vibrational signatures associated with such induced molecular fitting for two classes of transcription factors-the basic leucine-zipper (bZIP) motif and the high-mobility-group (HMG) box-each with a DNA target site. Results for bZIP are described here; findings for the HMG-box are reported in the preceding paper in this issue [Benevides, J. M., Chan, G., Lu, X.-J., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 537-547]. The yeast activator GCN4 provides a well-studied example of bZIP recognition, wherein B-DNA serves essentially as a template for protein folding. Analysis of Raman spectra of the 57-residue GCN4 bZIP domain, its AP-1 binding site, and their specific complex confirms a DNA-induced increase in alpha-helicity, attributable to folding of GCN4 basic arms with virtually no change in B-DNA structure, consistent with previous X-ray and NMR structure determinations. The absence of DNA perturbations in the bZIP model contrasts sharply with the HMG box, where DNA structure perturbations predominate. The bZIP and HMG-box models represent two opposing extremes in a range of induced fits identifiable by Raman spectroscopy. Previously characterized lambda repressor/operator complexes [Benevides, J. M., Weiss, M. A., and Thomas, G. J. (1994) J. Biol. Chem. 269, 10869-10878] occupy an intermediate position within this range. A comprehensive tabulation of Raman markers proposed as diagnostic of different protein/DNA recognition motifs is presented. The results are analyzed in terms of available DNA crystal structures (Nucleic Acid Database) to identify details of DNA conformation that correlate with specific Raman recognition markers.

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.

Molecular mechanism of DNA recognition by the alpha subunit of the Oxytricha telomere binding protein.

Interactions between telomeric DNA and the alpha subunit of the heterodimeric telomere binding protein of Oxytricha nova have been probed by Raman spectroscopy, CD spectroscopy, and nondenaturing gel electrophoresis. Telomeric sequences investigated include the Oxytricha 3' overhang, d(T4G4)2, and the related sequence dT6(T4G4)2, which incorporates a 5'-thymidylate leader. Corresponding nontelomeric isomers, d(TG)8 and dT6(TG)8, have also been investigated. Both d(T4G4)2 and dT6(T4G4)2 form stable hairpins that contain Hoogsteen G.G base pairs [Laporte, L., and Thomas, G. J., Jr. (1998) J. Mol. Biol. 281, 261-270]. The alpha subunit binds specifically and stoichiometrically to the dT6(T4G4)2 hairpin and alters its secondary structure by inducing conformational changes in the 5'-thymidylate leader without extensive disruption of G.G base pairing. Conversely, binding of the alpha subunit to d(T4G4)2 eliminates G.G pairing and unfolds the hairpin. DNA unfolding is accompanied by conformational changes affecting both the backbone and dG residues, as evidenced by Raman and CD spectra. Interestingly, the alpha subunit also forms complexes with the nontelomeric isomers, d(TG)8 and dT6(TG)8, evidenced by altered electrophoretic mobility in nondenaturing gels; however, Raman and CD spectra of complexes of the alpha subunit with nontelomeric DNA suggest no significant changes in backbone or deoxynucleoside conformations. Similarly, the alpha subunit binds to but does not appreciably alter the secondary structure of duplex DNA. The present results show that while the alpha subunit has the capacity to bind to Watson-Crick and different non-Watson-Crick motifs, DNA refolding is specific to the Oxytricha telomeric hairpin and the retention of G.G pairing is specific to the telomeric sequence incorporating the 5' leading sequence. A model is proposed for alpha subunit binding to telomeric DNA, and the physiological role of the alpha subunit in telomere organization is discussed.

Structural polymorphism and raman conformation markers of cyclic deoxytriadenylic acid.

X-ray analysis of two different trigonal crystal forms (space groups R32 and P3 ) of cyclic deoxytriadenylic acid [c(dAp)3] indicates for each an asymmetric unit consisting of two conformationally similar c(dAp)3molecules. Raman spectroscopy supports the X-ray interpretation for the R32 crystal, but identifies another c(dAp)3conformation not revealed in the P3 X-ray structure. The results for the P3 crystal can be explained if an additional c(dAp)3conformer is present but not sufficiently ordered within the lattice to contribute to X-ray diffraction. The Raman signature of aqueous c(dAp)3, which differs from signatures of both the R32 and P3 crystals, exhibits backbone markers similar to those of thermally denatured DNA and indicates that c(dAp)3molecules in solution populate a wider range of phosphoester ring conformations than in R32 and P3 crystals. Thus, polymorphism is observed for both crystal and solution structures of c(dAp)3. The results imply a highly flexible phosphoester ring that may be relevant to the function of cyclic oligonucleotides as biological effectors. A novel Raman marker at 821 cm-1is demonstrated as diagnostic of phosphoester torsions alpha and zeta in the gauche+ range. Specific Raman markers are also identified for the S -type (C2'- endo) deoxyadenosine conformations that occur in R32 and P3 crystal structures of c(dAp)3.

Polarized Raman spectroscopy of double-stranded RNA from bacteriophage phi6: local Raman tensors of base and backbone vibrations.

Raman tensors for localized vibrations of base (A, U, G, and C), ribose and phosphate groups of double-stranded RNA have been determined from polarized Raman measurements on oriented fibers of the genomic RNA of bacteriophage phi6. Polarized Raman intensities for which electric vectors of both the incident and scattered light are polarized either perpendicular (I[bb]) or parallel (I[cc]) to the RNA fiber axis have been obtained by Raman microspectroscopy using 514.5-nm excitation. Similarly, the polarized Raman components, I(bc) and I(cb), for which incident and scattered vectors are mutually perpendicular, have been obtained. Spectra collected from fibers maintained at constant relative humidity in both H2O and D2O environments indicate the effects of hydrogen-isotopic shifts on the Raman polarizations and tensors. Novel findings are the following: 1) the intense Raman band at 813 cm(-1), which is assigned to phosphodiester (OPO) symmetrical stretching and represents the key marker of the A conformation of double-stranded RNA, is characterized by a moderately anisotropic Raman tensor; 2) the prominent RNA band at 1101 cm(-1), which is assigned to phosphodioxy (PO2-) symmetrical stretching, also exhibits a moderately anisotropic Raman tensor. Comparison with results obtained previously on A, B, and Z DNA suggests that tensors for localized vibrations of backbone phosphodiester and phosphodioxy groups are sensitive to helix secondary structure and local phosphate group environment; and 3) highly anisotropic Raman tensors have been found for prominent and well-resolved Raman markers of all four bases of the RNA duplex. These enable the use of polarized Raman spectroscopy for the determination of purine and pyrimidine base residue orientations in ribonucleoprotein assemblies. The present determination of Raman tensors for dsRNA is comprehensive and accurate. Unambiguous tensors have been deduced for virtually all local vibrational modes of the 300-1800 cm(-1) spectral interval. The results provide a reliable basis for future evaluations of the effects of base pairing, base stacking, and sequence context on the polarized Raman spectra of nucleic acids.

DNA melting investigated by differential scanning calorimetry and Raman spectroscopy.

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.

Raman spectroscopy of the Ff gene V protein and complexes with poly(dA): nonspecific DNA recognition and binding.

Raman spectra of crystals and solutions of the single-stranded DNA binding protein of bacteriophage Ff (gene V protein, gVp) and of solution complexes of gVp with single-stranded poly-(deoxyadenylic acid) [poly(dA)] reveal the following: (i) The gVp secondary and tertiary structures are similar in solution and in the crystal and are dominated by beta-sheet domains, in agreement with NMR and X-ray findings. (ii) Subunit conformation and side chain environments of gVp are virtually unchanged over a wide range of salt concentration (0 < [NaCl] < 100 mM); however, the solution conformation of poly(dA) exhibits sensitivity to added salt. The perturbed Raman markers indicate subtle changes in helix backbone geometry with accompanying small differences in base stacking as the concentration of NaCl is changed. (iii) In complexes with poly(dA), neither the conformation of gVp nor its side chain environments are altered significantly in comparison to the free protein. This is the case at both high salt (nucleotide-to-subunit binding stoichiometry n = 4) and low salt (n = 3). (iv) The Raman signature of poly(dA) undergoes small perturbations upon gVp binding, indicative of small changes in base stacking and phosphodiester backbone conformation. The present results show that the different stoichiometric binding modes of gVp to poly(dA) are accomplished without significant changes in gVp subunit structure and with only modest changes in the single-stranded poly(dA) ligand. This contrasts sharply with sequence-specific double-stranded DNA binding proteins, such as the phage lambda and D108 repressors, which undergo substantial structural changes upon DNA binding, and which also alter more dramatically the Raman fingerprints of their DNA target sites. Thus, nonspecific and specific nucleic acid recognition modes are distinguishable by Raman spectroscopy. The Raman signature of gVp also allows examination of hydrogen bonding interactions of unique side chains within the hydrophobic core (cysteine 33) and at the binding interface (tyrosine 41). These are discussed in relation to the recently published gVp crystal structure.

Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures of DNA deoxycytidylates d(CCCT) and d(C8).

The Raman spectral signature of the four-stranded cytosine structure formed by intercalation of two hemiprotonated and parallel-stranded oligodeoxycytidylate duplexes (so-called i motif) has been obtained from the crystal structure of d(CCCT) [Kang, C.H., Berger, I., Lockshin, C., Ratliff, R., Moyzis, R., & Rich, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11636-11640]. Identification of Raman markers diagnostic of the cytosine quadruplex is complemented by results obtained in a pH titration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that the spectral fingerprint associated with N3 protonation of cytosine is distinct from that of quadruplex formation. The Raman spectrum thus provides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 protonation in DNA. Application to aqueous d(CCCT) and d(C8) demonstrates that the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and the d(CCCT) quadruplex exhibit a midpoint of titration (apparent pKc) of 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C8) is shifted significantly toward the physiological range, with pKc = 5.8 +/- 0.2. The difference in pKc between the two quadruplexes is equivalent to a free energy difference of 1.7 kcal/mol at 10 degrees C. The present findings extend the library of Raman conformation markers to deoxycytidylate residues in the novel i quadruplex. The significance of these results for probing solution conformations of telomeric DNA sequences is also considered.

Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+.

Differential scanning calorimetry, laser Raman spectroscopy, optical densitometry, and pH potentiometry have been used to investigate DNA melting profiles in the presence of the chloride salts of Ba2+, Sr2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. Metal-DNA interactions have been observed for the molar ratio [M2+]/[PO2-] = 0.6 in aqueous solutions containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. All of the alkaline earth metals, plus Mn2+, elevate the melting temperature of DNA (Tm > 75.5 degrees C), whereas the transition metals Co2+, Ni2+, and Cd2+ lower Tm. Calorimetric (delta Hcal) and van't Hoff (delta HVH) enthalpies of melting range from 6.2-8.7 kcal/mol bp and 75.6-188.6 kcal/mol cooperative unit, respectively, and entropies from 17.5 to 24.7 cal/K mol bp. The average number of base pairs in a cooperative melting unit () varied from 11.3 to 28.1. No dichotomy was observed between alkaline earth and transition DNA-metal complexes for any of the thermodynamic parameters other than their effects on Tm. These results complement Raman difference spectra, which reveal decreases in backbone order, base unstacking, distortion of glycosyl torsion angles, and rupture of hydrogen bonds, which occur after thermal denaturation. Raman difference spectroscopy shows that transition metals interact with the N7 atom of guanine in duplex DNA. A broader range of interaction sites with single-stranded DNA includes ionic phosphates, the N1 and N7 atoms of purines, and the N3 atom of pyrimidines. For alkaline earth metals, very little interaction was observed with duplex DNA, whereas spectra of single-stranded complexes are very similar to those of melted DNA without metal. However, difference spectra reveal some metal-specific perturbations at 1092 cm-1 (nPO2-), 1258 cm-1 (dC, dA), and 1668 cm-1 (nC==O, dNH2 dT, dG, dC). Increased spectral intensity could also be observed near 1335 cm-1 (dA, dG) for CaDNA. Optical densitometry, employed to detect DNA aggregation, reveals increased turbidity during the melting transition for all divalent DNA-metal complexes, except SrDNA and BaDNA. Turbidity was not observed for DNA in the absence of metal. A correlation was made between DNA melting, aggregation, and the ratio of Raman intensities I1335/I1374. At room temperature, DNA-metal interactions result in a pH drop of 1.2-2.2 units for alkaline earths and more than 2.5 units for transition metals. Sr2+, Ba2+, and Mg2+ cause protonated sites on the DNA to become thermally labile. These results lead to a model that describes DNA aggregation and denaturation during heating in the presence of divalent metal cations; 1) The cations initially interact with the DNA at phosphate and/or base sites, resulting in proton displacement. 2) A combination of metal-base interactions and heating disrupts the base pairing within the DNA duplex. This allows divalent metals and protons to bind to additional sites on the DNA bases during the aggregation/melting process. 3) Strands whose bases have swung open upon disruption are linked to neighboring strands by metal ion bridges. 4) Near the midpoint of the melting transition, thermal energy breaks up the aggregate. We have no evidence to indicate whether metal ion cross-bridges or direct base-base interactions rupture first. 5) Finally, all cross-links break, resulting in single-stranded DNA complexed with metal ions.

A phase diagram for sodium and potassium ion control of polymorphism in telomeric DNA.

Switching between antiparallel and parallel quadruplex structures of telomeric DNA under the control of intracellular Na+ and K+ has been implicated in the pairing of chromosomes during meiosis. Using Raman spectroscopy, we have determined the dependence of the interquadruplex equilibrium of the telomeric repeat of Oxytricha nova, upon solution concentrations of Na+ and K+. Both alkali cations facilitate the formation of an antiparallel foldback quadruplex at low concentration, and a parallel extended quadruplex at higher concentration. However, K+ is more effective than Na+ in inducing the parallel association. We propose a phase diagram relating d(T4G4)4 polymorphism to intracellular [Na+]/[K+] ratios. The phase diagram indicates that the interquadruplex equilibrium is highly sensitive to changes in the mole fraction of either cation when the total concentration falls within the interval 65 to 225 mM, a range which encompasses total of the Na+ and K+ concentrations occurring in a typical mammalian cell. These results support a role for the guanine-rich overhang of eukaryotic DNA in promoting chromosome association during meiotic synapsis.

Polarized Raman spectra of oriented fibers of A DNA and B DNA: anisotropic and isotropic local Raman tensors of base and backbone vibrations.

Polarized Raman spectra of oriented fibers of calf thymus DNA in the A and B conformations have been obtained by use of a Raman microscope operating in the 180 degrees back-scattering geometry. The following polarized Raman intensities in the spectral interval 200-1800 cm-1 were measured with both 514.5 and 488.0 nm laser excitations: (1) Icc, in which the incident and scattered light are polarized parallel to the DNA helical axis (c axis); (2) Ibb, in which the incident and scattered light are polarized perpendicular to c; and (3) Ibc and Icb, in which the incident and scattered light are polarized in mutually perpendicular directions. High degrees of structural homogeneity and unidirectional orientation were confirmed for both the A and B form fibers, as judged by comparison of the observed Raman markers and intensity anisotropies with measurements reported previously for oligonucleotide single crystals of known three-dimensional structures. The fiber Raman anisotropies have been combined with solution Raman depolarization ratios to evaluate the local tensors corresponding to key conformation-sensitive Raman bands of the DNA bases and sugar-phosphate backbone. The present study yields novel vibrational assignments for both A DNA and BDNA conformers and also confirms many previously proposed Raman vibrational assignments. Among the significant new findings are the demonstration of complex patterns of A form and B form indicator bands in the spectral intervals 750-900 and 1050-1100 cm-1, the identification of highly anisotropic tensors corresponding to vibrations of base, deoxyribose, and phosphate moieties, and the determination of relatively isotropic Raman tensors for the symmetrical stretching mode of phosphodioxy groups in A and B DNA. The present fiber results provide a basis for exploitation of polarized Raman spectroscopy to determine DNA helix orientation as well as to probe specific nucleotide residue orientations in nucleoproteins, viruses, and other complex biological assemblies.

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: evidence for altered protein and DNA structures in the complex.

Ner repressors of the transposable phages Mu and D108 play a central role in regulating the expression of the early (transposase) operon and in ensuring that phage growth proceeds along a lytic pathway. The latter function is analogous to that performed by the Cro protein of phage lambda. Unlike lambda Cro, however, the structural basis of operator recognition is not known for the Ner repressors. In order to elucidate the structural features underlying operator recognition by Ner repressors, we have employed Raman spectroscopy as a probe of the solution secondary structures of both D108 Ner and Mu Ner. Additionally, we have obtained Raman spectra of the D108 Ner repressor when bound to a 61-base-pair oligodeoxynucleotide containing the 55-base-pair D108 ner binding site. Conformation-sensitive Raman bands show that both D108 and Mu Ner contain similar, highly alpha-helical (approximately 45%) secondary structures. The Raman markers also show that the substantial nonhelical secondary structure of both D108 Ner and Mu Ner is largely beta-stranded. The protein-free 61-bp D108 ner operator exhibits Raman marker bands diagnostic of an uninterrupted B DNA duplex. In the D108 Ner:DNA complex, we find the following: (i) B DNA stereochemistry is fully conserved, although with significant perturbations to the B form backbone geometry, particularly in AT-rich regions of the bound operator. (ii) The specific interactions that occur between Ner repressor and operator involve B DNA major groove sites. (iii) A small (8 +/- 3%) increase in alpha-helix content of the Ner repressor is detected upon operator binding. (iv) Finally, the local environments of many aromatic amino acids are substantially altered in the D108 Ner:DNA complex. We propose a molecular model for binding of D108 Ner to its operator that is consistent with both the present spectroscopic findings and the results of recent biochemical studies. Essential features of this model are bending of the DNA double helix and contact of operator sites with repressor domains bearing sequence homologies with the helix-turn-helix (HTH) motifs of other DNA-binding proteins. The Raman fingerprint of the Ner:DNA complex is shown to be clearly distinguishable from that of the lambda cI:DNA complex, even though both gene regulatory complexes are presumed to employ HTH recognition motifs. The unique Raman signatures observed for these repressor complexes suggest that the Raman methodology may be useful in discriminating different modes of operator recognition by the HTH motifs of regulatory proteins.

An altered specificity mutation in the lambda repressor induces global reorganization of the protein-DNA interface.

The lambda repressor exhibits structural characteristics of lock and key complementary through the helix-turn-helix motif, and of induced fit by virtue of DNA-dependent folding of the N-terminal arm. In both cases, molecular recognition is mediated by direct contacts between amino acids and DNA bases. The extent to which such contacts function as discrete elements in a protein-DNA recognition code is not known. Because of the relevance of protein recognition to the broader issue of protein design, and because the lambda system serves as a prototype for gene regulation, we have employed laser Raman and 1H NMR spectroscopy to compare free and operator-bound structures of lambda repressor variants which are known to exhibit altered DNA-binding specificities. Experimental design is based upon a previous biochemical study of mutations in the repressor N-terminal arm (K4Q) and helix-turn-helix motif (G48S) (Nelson, H. C. M., and Sauer, R. T. (1986) J. Mol. Biol. 192, 27-38). These mutations, which were originally isolated by loss of function (K4Q) and second-site reversion (G48S), are of particular interest in light of their complex effects on sequence specificity at multiple positions in the operator site (Benson, N., Adams, C., and Youderian, P. (1992) Genetics 130, 17-26). Laser Raman and 1H NMR spectra of repressor variants carrying one (G48S) or two mutations (K4Q/G48S) are similar to those of the native wild type repressor and are in accord with the x-ray crystal structure. Remarkably, however, the complexes of wild type and mutant repressors exhibit extensive differences both in the global DNA structure and in the environments of key functional groups along the major groove. By demonstrating that single amino acid substitutions can induce global reorganization of a protein-DNA interface, the present results establish that repressor-operator recognition in solution cannot be explained in terms of a simple recognition code.

Demonstration of Z-d(5BrCGAT5BrCG) and B-d(CGCGATCGCG) form crystal structures in DNA-cobalt hexammine complexes by Kr 647.1 nm excitation of Raman spectra.

Cobalt hexammine [Co(NH3)6(3+)] is an efficient DNA complexing agent which significantly perturbs nucleic acid secondary structure. We have employed red excitation (647.1 nm) from a krypton laser to obtain Raman spectra of the highly colored complexes formed between cobalt hexammine and crystals of the DNA oligomers, d(5BrCGAT5BrCG) and d(CGCGATCGCG), both of which incorporate out-of-alternation pyrimidine/purine sequences. The Co(NH3)6(3+) complex of d(5BrCGAT5BrCG) exhibits a typical Z-form Raman signature, similar to that reported previously for the alternating d(CGCGCG) sequence. Comparison of the Raman bands of d(5BrCGAT5BrCG) with those of other oligonucleotide and polynucleotide structures suggests that C3'-endo/syn and C3'-endo/anti thymidines may exhibit distinctive nucleoside conformation markers, and tentative assignments are proposed. The Raman markers for C2'-endo/anti adenosine in this Z-DNA are consistent with those reported previously for B-DNA crystals containing C2'-endo/anti dA. Raman bands of the cobalt hexammine complex of d(CGCGATCGCG) are those of B-DNA, but with significant differences from the previously characterized B-DNA dodecamer, d(CGCAAATTTGCG). The observed differences suggest an unusual deoxyguanosine conformer, possibly related to a previously characterized structural intermediate in the B-->Z transition. The present results show that crystallization of d(CGCGATCGCG) in the presence of cobalt hexammine is not alone sufficient to induce the left-handed Z-DNA conformation. This investigation represents the first application of off-resonance Raman spectroscopy for characterization of highly chromophoric DNA and illustrates the feasibility of the Raman method for investigating other structurally perturbed states of DNA-cobalt hexammine complexes.

DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage lambda repressor and its interaction with operator sites OL1 and OR3.

The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.

Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser Raman spectroscopy.

The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)