Biomedical subjects

James M Benevides

Publications and source records attributed to James M Benevides.

12 recordsLinked to original sources

PubMed2006

The complex of ethidium bromide with genomic DNA: structure analysis by polarized Raman spectroscopy.

Structural properties of the complex formed between genomic DNA and the intercalating drug ethidium bromide (EtBr) have been determined by use of a Raman microscope equipped with near-infrared laser excitation. The polarized spectra, which were obtained from oriented fibers of the EtBr:DNA complex, are interpreted in terms of the relative orientations of the phenanthridinium ring of EtBr and bases of DNA. Quantification of structure parameters of EtBr and DNA in the complex were assessed using Raman tensors obtained from polarized Raman analyses of oriented specimens of EtBr (single crystal) and DNA (hydrated fiber). We find that the phenanthridinium plane is tilted by 35+/-5 degrees from the plane perpendicular to the fiber (DNA helix) axis. Assuming coplanarity of the phenanthridinium ring and its immediate base neighbors at the intercalation site, such bases would have a tilt angle closer to that of A-DNA (20 degrees) than to that of B-DNA (6 degrees). The average base tilt in stretches of DNA between intercalation sites remains that of B-DNA.

Binding Sites↗

PubMed2006

Structural perturbations induced in linear and circular DNA by the architectural protein HU from Bacillus stearothermophilus.

HU is a small DNA-binding protein of eubacteria that is believed to induce or stabilize bending of the double helix and mediate nucleoid compaction in vivo. Although HU does not bind preferentially to specific DNA sequences, it is known to have high affinity for DNA sites containing structural anomalies, such as unpaired or mismatched bases, nicks, and four-way junctions. We have employed Raman spectroscopy to further investigate the structural basis of HU-DNA recognition in solution. Experiments were carried out on the homodimeric HU protein of Bacillus stearothermophilus (HUBst) and a 222-bp DNA fragment, which was isolated in linear (DNA(L222)) and circular (DNA(C222)) forms. In the absence of bound HUBst the Raman signatures of DNA(L222) and DNA(C222) are nearly superimposable, indicating that circularization produces no substantial change in the local B-DNA conformation. Conversely, the Raman signatures of DNA(L222) and DNA(C222) are perturbed significantly and specifically by HUBst binding. The HUBst-induced perturbations are markedly greater for the circularized DNA target. These results support an opportunistic molecular mechanism, in which HU binding is facilitated by intrinsic nonlinearity or flexibility in the DNA target. We propose that DNA segments which are bent or predisposed toward bending provide the high-affinity sites for HU attachment and nucleoid condensation. This model is consistent with the wide range of DNA bending angles reported in crystal structures of HU-DNA complexes.

Bacterial Proteins↗

PubMed2005

Structural details of the thermophilic filamentous bacteriophage PH75 determined by polarized Raman microspectroscopy.

The filamentous virus PH75, which infects the thermophile Thermus thermophilus, consists of a closed DNA strand of 6500 nucleotides encapsidated by 2700 copies of a 46-residue coat subunit (pVIII). The PH75 virion is similar in composition to filamentous viruses infecting mesophilic bacteria but is distinguished by in vivo assembly at 70 degrees C and thermostability to at least 90 degrees C. Structural details of the PH75 assembly are not known, although a fiber X-ray diffraction based model suggests that capsid subunits are highly alpha-helical and organized with the same symmetry (class II) as in the mesophilic filamentous phages Pf1 and Pf3 [Pederson et al. (2001) J. Mol. Biol. 309, 401-421]. This is distinct from the symmetry (class I) of phages fd and M13. We have employed polarized Raman microspectroscopy to obtain further details of PH75 architecture. The spectra are interpreted in combination with known Raman tensors for modes of the pVIII main chain (amide I) and Trp and Tyr side chains to reveal the following structural features of PH75: (i) The average pVIII peptide group is oriented with greater displacement from the virion axis than peptide groups of fd, Pf1, or Pf3. The data correspond to an average helix tilt angle of 25 degrees in PH75 vs 16 degrees in fd, Pf1, and Pf3. (ii) The indolyl ring of Trp 37 in PH75 projects nearly equatorially from the subunit alpha-helix axis, in contrast to the more axial orientations for Trp 26 of fd and Trp 38 of Pf3. (iii) The phenolic rings of Tyr 15 and Tyr 39 project along the subunit helix axis, and one phenoxyl engages in hydrogen-bonding interaction that has no counterpart in either fd or Pf1 tyrosines. Also, in contrast to fd, Pf1, and Pf3, the packaged DNA genome of PH75 exhibits no Raman anisotropy, suggesting that DNA bases are not oriented unidirectionally within the nucleocapsid assembly. The structural findings are discussed in relation to intrasubunit and intersubunit interactions that may confer hyperthermostability to the PH75 virion. A refined molecular model is proposed for the PH75 capsid subunit.

Amides↗

PubMed2005

Local conformational changes induced in B-DNA by ethidium intercalation.

Structural effects of binding the intercalating drug ethidium bromide (EtBr) to 160 base pair (bp) fragments of nucleosomal calf thymus DNA have been probed by the method of Raman difference spectroscopy. With the use of a near-infrared (NIR) laser source to excite the Raman spectrum at 752 nm, vibrational signatures of both the EtBr intercalant and DNA target have been identified in spectra of the drug-DNA complexes. Analysis of the results obtained on complexes consisting of 1 EtBr bound/10 bp leads to the following conclusions: (i) Raman markers diagnostic of DNA phosphodiester conformation are converted from the B type to the A type with EtBr binding, commensurate with the proportion of ethidium-bound nucleotides in the complex. (ii) Ethidium binding converts deoxynucleoside sugar puckers from the C2'-endo to the C3'-endo conformation, also consistent with binding stoichiometry. Both pyrimidine and purine deoxynucleoside sugar puckers are perturbed by the phenanthridinium ring intercalation. (iii) Phenanthridinium insertion between bases is accomplished with no apparent change in hypochromicities of purine or pyrimidine Raman markers, indicating that base-phenanthridinium interactions provide compensatory hypochromic effects. (iv) Novel Raman markers of helix unwinding have been identified and assigned primarily to methylene deformation modes of the deoxyribosyl C2'H(2) and C5'H(2) groups. The present study provides new insights into drug-DNA recognition in solution and demonstrates the feasibility of NIR-Raman spectroscopy for structural studies of highly chromophoric DNA complexes.

Animals↗

PubMed2004

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 A 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 A 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.

Bacteriophage PRD1↗

PubMed2004

Domain structures and roles in bacteriophage HK97 capsid assembly and maturation.

Head assembly in the double-stranded DNA coliphage HK97 involves initially the formation of the precursor shell Prohead I from approximately 420 copies of a 384-residue subunit. This is followed by proteolytic removal of residues 2-103 to create Prohead II, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make Head II. Here, we report and structurally interpret solution Raman spectra of Prohead I, Prohead II, and Head II particles. The Raman signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --> Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --> capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures.

Asparagine↗

PubMed2003

HU protein employs similar mechanisms of minor-groove recognition in binding to different B-DNA sites: demonstration by Raman spectroscopy.

The sequence isomers d(CGCAAATTTGCG) and d(TCAAGGCCTTGA) form self-complementary duplexes that present distinct targets for binding of the homodimeric architectural protein HU of Bacillus stearothermophilus (HUBst). Raman spectroscopy shows that although each duplex structure is of the B-DNA type, there are subtle conformational dissimilarities between them, involving torsion angles of the phosphodiester backbone and the arrangements of stacked bases. Each DNA duplex forms a stable stoichiometric (1:1) complex with HUBst, in which the structure of the HUBst dimer is largely conserved. However, the Raman signature of each DNA duplex is perturbed significantly and similarly with HUBst binding, as reflected in marker bands assigned to localized vibrations of the phosphodiester moieties and base residues. The spectral perturbations identify a reorganization of the DNA backbone and partial unstacking of bases with HUBst binding, which is consistent with non-sequence-specific minor-groove recognition. Prominent among the HUBst-induced perturbations of B-DNA are a conversion of approximately one-third of the alpha/beta/gamma torsions from the canonical g(-)/t/g(+) conformation to an alternative conformation, an equivalent conversion of deoxyadenosyl moieties from the C2'-endo/anti to the C3'-endo/anti conformation, and appreciable unstacking of purines. The results imply that each solution complex is characterized by structural perturbations extending throughout the 12-bp sequence. Comparison with previously studied protein/DNA complexes suggests that binding of HUBst bends DNA by approximately 70 degrees.

Bacterial Proteins↗

PubMed2002

Characterization of subunit-specific interactions in a double-stranded RNA virus: Raman difference spectroscopy of the phi6 procapsid.

The icosahedral core of a double-stranded (ds) RNA virus hosts RNA-dependent polymerase activity and provides the molecular machinery for RNA packaging. The stringent requirements of dsRNA metabolism may explain the similarities observed in core architecture among a broad spectrum of dsRNA viruses, from the mammalian rotaviruses to the Pseudomonas bacteriophage phi6. Although the structure of the assembled core has been described in atomic detail for Reoviridae (blue tongue virus and reovirus), the molecular mechanism of assembly has not been characterized in terms of conformational changes and key interactions of protein constituents. In the present study, we address such questions through the application of Raman spectroscopy to an in vitro core assembly system--the procapsid of phi6. The phi6 procapsid, which comprises multiple copies of viral proteins P1 (copy number 120), P2 (12), P4 (72), and P7 (60), represents a precursor of the core that is devoid of RNA. Raman signatures of the procapsid, its purified recombinant core protein components, and purified sub-assemblies lacking either one or two of the protein components have been obtained and interpreted. The major procapsid protein (P1), which forms the skeletal frame of the core, is an elongated and monomeric molecule of high alpha-helical content. The fold of the core RNA polymerase (P2) is also mostly alpha-helical. On the other hand, the folds of both the procapsid accessory protein (P7) and RNA-packaging ATPase (P4) are of the alpha/beta type. Raman difference spectra show that conformational changes occur upon interaction of P1 with either P4 or P7 in the procapsid. These changes involve substantial ordering of the polypeptide backbone. Conversely, conformations of procapsid subunits are not significantly affected by interactions with P2. An assembly model is proposed in which P1 induces alpha-helix in P4 during formation of the nucleation complex. Subsequently, the partially disordered P7 subunit is folded within the context of the procapsid shell.

Bacteriophage phi 6↗

PubMed2002

Secondary structure polymorphism in Oxytricha nova telomeric DNA.

Tandem repeats of the telomeric DNA sequence d(T4G4) of Oxytricha nova are capable of forming unusually stable secondary structures incorporating Hoogsteen hydrogen bonding interactions. The biological significance of such DNA structures is supported by evidence of specific recognition of telomere end-binding proteins in the crystal state. To further characterize structural polymorphism of Oxytricha telomeric DNAs, we have obtained and interpreted Raman, ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectra of the tandem repeats d(G4T4G4) (Oxy1.5), d(T4G4)2 (Oxy2) and dT6(T4G4)2 (T6Oxy2) and related non-telomeric isomers in aqueous salt solutions. Raman markers of Oxy1.5 identify both C2'-endo/anti and C2'-endo/syn conformations of the deoxyguanosine residues and Hoogsteen hydrogen bonded guanine quartets, consistent with the quadruplex fold determined previously by solution NMR spectroscopy. Raman, UVRR and CD signatures and Raman dynamic measurements, to monitor imino NH-->ND exchanges, show that the Oxy1.5 antiparallel quadruplex fold is distinct from the hairpin structures of Oxy2 and T6Oxy2, single-stranded structures of d(TG)8 and dT6(TG)8 and previously reported quadruplex structures of d(T4G4)4 (Oxy4) and dG12. Spectral markers of the telomeric and telomere-related DNA structures are tabulated and novel Raman and UVRR indicators of thymidine and deoxyguanosine conformations are identified. The results will be useful for probing structures of Oxytricha telomeric repeats in complexes with telomere end-binding proteins.

Animals↗

PubMed2002

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA.

In previous papers of this series the temperature-dependent Raman spectra of poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A.T and alternating A.T/T.A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA).poly(dT) and poly(dA-dT). poly(dA-dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van't Hoff premelting enthalpies of poly(dA).poly(dT) [DeltaH(vH)(pm) = 18.0 +/- 1.6 kcal x mol(-1) (kilocalories per mole cooperative unit)] and poly(dA-dT).poly(dA-dT) (DeltaH(vH)(pm) = 13.4 +/- 2.5 kcal x mol(-1)) differ by an amount (approximately 4.6 kcal x mol(-1)) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)(n).(dT)(n) tracts. (iii) The overall stacking free energy of poly(dA). poly(dT) [-6.88 kcal x mol(bp)(-1) (kilocalories per mole base pair)] is greater than that of poly(dA-dT). poly(dA-dT) (-6.31 kcal x mol(bp)(-1)). (iv) The difference between stacking free energies of A and T is significant in poly(dA).poly(dT) (DeltaDeltaG(st) = 0.8 +/- 0.3 kcal. mol(bp)(-1)), but marginal in poly(dA-dT).poly(dA-dT) (DeltaDeltaG(st) = 0.3 +/- 0.3 kcal x mol(bp)(-1)). (v) In poly(dA). poly(dT), the van't Hoff parameters for melting of A (DeltaH(vH)(A) = 407 +/- 23 kcal.mol(-1), DeltaS(vH)(A) = 1166 +/- 67 cal. degrees K(-1) x mol(-1), DeltaG(vH(25 degrees C))(A) = 60.0 +/- 3.2 kcal x mol(-1)) are clearly distinguished from those of T (DeltaH(vH)(T) = 185 +/- 38 kcal x mol(-1), DeltaS(vH)(T) = 516 +/- 109 cal. degrees K(-1) x mol(-1), DeltaG(vH(25 degrees C))(T) = 27.1 +/- 5.5 kcal x mol(-1)). (vi) Similar relative differences are observed in poly(dA-dT). poly(dA-dT) (DeltaH(vH)(A) = 333 +/- 54 kcal x mol(-1), DeltaS(vH)(A) = 961 +/- 157 cal. degrees K(-1) x mol(-1), DeltaG(vH(25 degrees C))(A) = 45.0 +/- 7.6 kcal x mol(-1); DeltaH(vH)(T) = 213 +/- 30 kcal x mol(-1), DeltaS(vH)(T) = 617 +/- 86 cal. degrees K(-1) x mol(-1), DeltaG(vH(25 degrees C))(T) = 29.3 +/- 4.9 kcal x mol(-1)). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.

Base Composition↗

PubMed2002

DNA secondary structure and Raman markers of supercoiling in Escherichia coli plasmid pUC19.

Negative supercoiling in the 2686 bp Escherichia coli plasmid pUC19 is comparable in linking number (Lk(0) = 258) and superhelical density (sigma = -0.05) to the moderate supercoiling exhibited by many eukaryotic chromosomal DNAs in vivo. Supercoiled and relaxed forms of purified pUC19 in aqueous solution (0.1 M NaCl, pH 8.3, 20 degrees C) have been investigated by Raman spectroscopy to assess changes in B-DNA secondary structure induced by superhelical stress and to identify putative Raman markers of DNA supercoiling. We find that supercoiling leads to small but significant changes to the B-form Raman signature of linear DNA. Spectral band shifts in the 780-850 cm(-1) interval are interpreted as resulting from a small net change in the average phosphodiester torsions alpha (O3'-P->-O5'-C5') and zeta (C3'-O3'->-P-O5') from the gauche(-)/gauche(-) range to the gauche(-)/trans range with supercoiling. The magnitude of the spectral intensity change implies that approximately 5% of the nucleotide moieties are affected. Supercoiling also introduces small redistributions of Raman intensity within the 1460-1490 and 1660-1670 cm(-1) intervals, consistent with small structural perturbations. Importantly, no Raman markers of Watson-Crick base pairing, base stacking, or C2'-endo/anti deoxynucleoside conformations are perturbed significantly by supercoiling of pUC19, indicating that the B-DNA structure is largely conserved under moderate superhelical stress. Peak and trough features at 814 and 783 cm(-1), and at 1462 and 1489 cm(-1), respectively, in the Raman difference spectrum between superhelical and relaxed DNA are proposed as markers of moderate negative supercoiling. We also show that in Tris-buffered solutions the Raman signature of supercoiled DNA can be obscured by Raman bands of Tris counterions. The subtle structural perturbations to B-DNA induced by moderate supercoiling are consistent with proposed mechanisms of transcriptional activation.

Base Sequence↗

PubMed2002

Temperature dependence of the Raman spectrum of DNA. II. Raman signatures of premelting and melting transitions of poly(dA).poly(dT) and comparison with poly(dA-dT).poly(dA-dT).

The temperature dependence of the Raman spectrum of poly(dA).poly(dT) (dA: deoxyadenosine; dT: thymidine), a model for DNA containing consecutive adenine.thymine (A.T) pairs, has been analyzed using a spectrometer of high spectral precision and sensitivity. Three temperature intervals are distinguished: (a) premelting (10 < t < 70 degrees C), in which the native double helix is structurally altered but not dissociated into single strands; (b) melting (70 < t < 80 degrees C), in which the duplex is dissociated into single strands; and (c) postmelting (80 < t degrees C), in which no significant structural change can be detected. The distinctive Raman difference signatures observed between 10 and 70 degrees C and between 70 and 80 degrees C are interpreted in terms of the structural changes specific to premelting and melting transitions, respectively. Premelting alters the low-temperature conformation of the deoxyribose-phosphate backbone and eliminates base hydrogen bonding that is distinct from canonical Watson-Crick hydrogen bonding; these premelting perturbations occur without disruption of base stacking. Conversely, melting eliminates canonical Watson-Crick pairing and base stacking. The results are compared with those reported previously on poly(dA-dT).poly(dA-dT), the DNA structure consisting of alternating A.T and T.A pairs (L. Movileanu, J. M. Benevides, and G. J. Thomas, Jr. Journal of Raman Spectroscopy, 1999, Vol. 30, pp. 637-649). Poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT) exhibit strikingly dissimilar temperature-dependent Raman profiles prior to the onset of melting. However, the two duplexes exhibit very similar melting transitions, including the same Raman indicators of ruptured Watson-Crick pairing, base unstacking and collapse of backbone order. A detailed analysis of the data provides a comprehensive Raman assignment scheme for adenosine and thymidine residues of B-DNA, delineates Raman markers diagnostic of consecutive A.T and alternating A.T/T.A tracts of DNA, and identifies the distinct Raman difference signatures for premelting and melting transitions in the two types of sequences.

Base Composition↗