James M. Benevides

Membrane structure and interactions with protein and DNA in bacteriophage PRD1

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 Å resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 Å resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.

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.

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.

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.

Temperature dependence of the Raman spectrum of DNA. Part I - Raman signatures of premelting and melting transitions of Poly(dA-dT)·poly(dA-dT)

Poly(dA–dT)· poly(dA–dT) is a double-helical B DNA containing A· T and T· A base pairs in alternating sequence. Although Raman spectra of this structure have been reported previously, the temperature dependence of the Raman bands has not been examined in detail. Using a spectrometer of high spectral precision and sensitivity, we applied Raman difference spectroscopy to determine the temperature dependence of all Raman bands of poly(dA–dT)· poly(dA–dT) in physiological salt solutions (both H2O and D2O) over the temperature range 10–85  °C. Three temperature domains are distinguished by Raman spectroscopy: premelting (10 < t < 66  °C), in which the double-stranded structure is perturbed but does not dissociate; melting (66 < t < 75  °C), in which the double-stranded structure dissociates; and postmelting (75 < t < 85  °C), in which no structural change can be detected. The results demonstrate that distinct Raman difference signatures exist for the premelting and melting transitions, and that each involves changes in most Raman bands. Among other novel findings and assignments: (i) Raman bands at 728 (dA), 1236 (dT) and 1301 cm−1 (dA) provide the most sensitive measures of base stacking in both the premelting and melting domains. (ii) Raman bands at 1182 (dT) and 1512 cm−1 (dA) provide the most sensitive measure of A· T unpairing in the melting domain. An unexpected finding is the apparent correlation of the 1182 cm−1 band intensity with the degree of thymine unpairing. Additionally, bands at 1376 (dT) and 1577 cm−1 (dA) confirm changes in interbase hydrogen bonding with premelting. (iii) The temperature dependence of the Raman band at 819 cm−1 is closely correlated with those of backbone marker bands at 792 and 842 cm−1 (O– P– O stretch modes), indicating that the 819 cm−1 band is due to a vibrational mode localized in the backbone and is diagnostic of phosphodiester torsions α / ζ apparently specific to the gauche−/trans conformation of BII DNA. (iv) The Raman band at 1484 cm−1 (dA) is sensitive to hydrogen bonding at the adenine N1 site. (ν) Comparison of spectra from H2O and D2O solutions reveal significant vibrational coupling between Raman bands of the deoxynucleoside residues and B-form backbone of poly(dA–dT)·poly(dA–dT). These results constitute an important foundation for future Raman studies of premelting and melting phenomena in other DNA sequences, including poly(dA)·poly(dT). Copyright

Raman Spectroscopy of Proteins

A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side ‐chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three‐dimensional structure, intramolecular dynamics, and intermolecular interactions. Here, the strengths of Raman spectroscopy are illustrated by considering recent applications that address (1) subunit folding and recognition in assembly of the icosahedral capsid of bacteriophage P22, (2) orientations of subunit main chains and side chains in native filamentous viruses, (3) roles of cysteine hydrogen bonding in the folding, assembly, and function of virus structural proteins, and (4) structural determinants of protein/DNA recognition in gene regulatory complexes. Conventional Raman, UV‐resonance Raman, and polarized Raman techniques are surveyed.

Secondary structure and thermostability of the phage P22 tailspike: XX. Analysis by Raman spectroscopy of the wild-type protein and a temperature-sensitive folding mutant

The thermostable tailspike endorhamnosidase of bacteriophage P22 has been investigated by laser Raman spectroscopy to determine the proteins secondary structure and the basis of its thermostability. The conformation of the native tailspike, determined by Raman amide I and amide III band analyses, is 52 to 61% beta-sheet, 24 to 27% alpha-helix, 15 to 21% beta-turn and 0 to 10% other structure types. The secondary structure of the wild-type tailspike, as monitored by the conformation-sensitive Raman amide bands, was stable to 80 degrees C, denatured reversibly between 80 and 90 degrees C, and irreversibly above 90 degrees C. The purified native form of a temperature-sensitive folding mutant (tsU38) contains secondary structures virtually identical to those in the wild-type in aqueous solution at physiological conditions (0.05 M-Na+ (pH 7.5], at both permissive (20 degrees C) and restrictive (40 degrees C) temperatures. This supports previous results showing that the mutational defect at 40 degrees C affects intermediates in the folding pathway rather than the native structure. At temperatures above 60 degrees C the wild-type and mutant forms were distinguishable: the reversible and irreversible denaturation thresholds were approximately 15 to 20 degrees C lower in the mutant than in the wild-type protein. The irreversible denaturation of the mutant tailspikes led to different aggregation/polymerization products from the wild-type, indicating that the mutation altered the unfolding pathway. In both cases only a small percentage of the native secondary structure was altered by irreversible thermal denaturation, indicating that the aggregated states retain considerable native structure.

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.

Solution conformations of nucleoside analogues exhibiting antiviral activity against human immunodeficiency virus

Abstract The molecular-conformational basis for HIV-1 antiviral activity of dideoxynucleoside analogues is unknown. A recent proposal by van Roey [1] that furanose sugar puckering in the C2′ -endo family (namely C3′ -exo) may account for the enhanced anti-HIV-1 activity of azidothymidine (AZT), dideoxythymidine (ddT) and dideoxycytidine (ddC) has been tested by conformational analysis of these and related agents, using laser Raman spectroscopy of their solutions and crystal structures. The results show that nucleoside analogues exhibiting anti-HIV-1 activity, including AZT, ddT and ddC, exist in solution with C3′ -endo as the predominating sugar pucker. The C3′ -endo solution conformations differ fundamentally from the C3′ -exo conformations observed in the corresponding crystal structures. Accordingly, the crystal conformation cannot be responsible for enhanced recognition of these agents, either by nucleoside kinase or reverse transcriptase, as a mechanism to explain antiviral activity. The present findings suggest that C3′ -endo sugear pucker, rather than C3′ -exo pucker, or other puckers of the C2′ -endo family, is more probably the required conformation for antivaral activity. The present work also shows that nucleoside phosphorylation does not, in general, change the preferred solution conformation of a nucleoside. Therefore, C3′ -endo sugar pucker is likely to be the preferred conformation for both nucleoside kinase and reverse transcriptase recognition. In this study, the list of thymidine nucleoside conformation markers available from Raman spectra is extended and additional group frequency assignments for C3′ -azido, C3′ -deoxy and related nucleoside derivatives are provided.

A Structural Model for the Single-Stranded DNA Genome of Filamentous Bacteriophage Pf1

The filamentous bacteriophage Pf1, which infects strain PAK of Pseudomonas aeruginosa, is a flexible filament ( approximately 2000 x 6.5 nm) consisting of a covalently closed DNA loop of 7349 nucleotides sheathed by 7350 copies of a 46-residue alpha-helical subunit. The subunit alpha-helices, which are inclined at a small average angle ( approximately 16 degrees ) from the virion axis, are arranged compactly around the DNA core. Orientations of the Pf1 DNA nucleotides with respect to the filament axis are not known. In this work we report and interpret the polarized Raman spectra of oriented Pf1 filaments. We demonstrate that the polarizations of DNA Raman band intensities establish that the nucleotide bases of packaged Pf1 DNA are well ordered within the virion and that the base planes are positioned close to parallel to the filament axis. The present results are combined with a previously proposed projection of the intraviral path of Pf1 DNA [Liu, D. J., and Day, L. A. (1994) Science 265, 671-674] to develop a novel molecular model for the Pf1 assembly.