Biomedical subjects

G J Thomas

Publications and source records attributed to G J Thomas.

At least 109 records · Page 6Linked to original sources

PubMed1994

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.

Amino Acid Sequence↗

PubMed1994

Novel tyrosine markers in Raman spectra of wild-type and mutant (Y21M and Y24M) Ff virions indicate unusual environments for coat protein phenoxyls.

The tyrosine side chain generates a pair of distinctive Raman bands--a Fermi doublet near 850 and 830 cm-1--with relative intensities diagnostic of hydrogen bonding states of the phenolic acceptor and donor atoms [Siamwiza et al. (1975) Biochemistry 14, 4870-4876]. This structural correlation has been tested extensively and is used widely as an indicator of tyrosine interactions in globular proteins and their assemblies. However, in Ff filamentous viruses (fd, f1, M13) the apparent Fermi doublet intensity ratio (I853/I826 approximately 4.0) is much greater than the maximum predicted or observed in other proteins. To understand this anomaly, we have reevaluated the basis for the Fermi doublet assignment in Ff. We report Raman spectra of site-specific mutants of Ff in which either one (Y21M and Y24M) or both (Y21F/Y24S) tyrosines of the coat protein subunit (pVIII) have been mutated. These Raman data, together with those obtained from Ff virions carrying residue-specific tyrosyl (Y-d4) and phenylalanyl (F-d5) deuterations in pVIII, demonstrate conclusively that the 853 and 826 cm-1 bands of Ff do not constitute a typical tyrosine Fermi doublet: The observed 826 cm-1 Raman band of Ff is due not to tyrosine but to phenylalanine residues of pVIII. The 853 cm-1 Raman band thus constitutes the first known example of a "tyrosine singlet" in the Raman spectrum of a protein. The implications of this finding for Ff virion structure and its relevance to tyrosine markers in other proteins are discussed.

Amino Acid Sequence↗

PubMed1994

A comparative study of translucent apical dentine in vital and non-vital human teeth.

Previous studies have shown that dentinal apical translucency increases with age. Although the mechanism by which apical translucency is produced is still uncertain, it has been suggested that it develops at a slower rate in non-vital teeth. In this investigation the amount and distribution of apical translucent dentine in a group of age-matched vital and non-vital teeth were compared. Fifty-five non-vital (root-filled) teeth and 49 vital teeth were used. Freshly extracted teeth were fixed in 2% glutaraldehyde solution, sectioned longitudinally in the buccolingual plane and ground to a thickness of 150 microns. Each section was photographed in polarized light at a standard magnification against a ruled scale background. Black-and-white prints of standard magnification were produced and translucent apical dentine was plotted using an Apple II digitizer. Variables measured were length, length as a percentage of root length, area and area as a percentage of root area. Data were related to age and tooth type and analysed by Mann-Whitney and regression analysis. Significant differences (p < 0.05) were present when tooth types were pooled. There was more apical translucency present in all types of non-vital teeth when compared with vital teeth at any given age.

Adolescent↗

PubMed1994

Vibrational analysis of nucleic acids. I. The phosphodiester group in dimethyl phosphate model compounds: (CH3O)2PO2-, (CD3O)2PO2-, and (13CH3O)2PO2-.

Normal coordinate analyses and vibrational assignments are presented for the dimethyl phosphate anion [(CH3O)2PO2-] and its deuteriomethyl [(CD3O)2PO2-] and carbon-13 [(13CH3O)2PO2-] derivatives in the gauche-gauche conformation. The dimethyl phosphate anion, which is the simplest model for the nucleic acid phosphodiester moiety, exhibits many of the spectral complexities of DNA and RNA and has previously resisted a complete and consistent vibrational analysis. In the present study we make use of new experimental data on the dimethyl phosphate isotopomers, including Raman depolarization measurements, to develop a consistent valence force field for normal modes of the C--O--P--O--C phosphodiester network and its hydrogenic substituents, as well as for stretching and bending modes of the O--P--O network of the anionic phosphodioxy group (PO2-). The force field established for dimethyl phosphate incorporates one significant nonbonded force constant, introduced from ab initio calculations, to account for interaction between the two ester C--O bonds. This study resolves previous problematic assignments for conformation-sensitive symmetric (in-phase) and asymmetric (out-of-phase) skeletal stretching modes of the ester linkages and demonstrates substantial anharmonicity in the hydrogen-stretching vibrations of the methyl substituents. New assignments are proposed for Raman bands of the phosphodioxy group, which may serve as potential indicators of structure and interaction of the DNA phosphates.

Carbon Isotopes↗

PubMed1993

Determination of the pKa values of active-center cysteines, cysteines-32 and -35, in Escherichia coli thioredoxin by Raman spectroscopy.

We have determined the pKa values for thiol-thiolate equilibria of Escherichia coli thioredoxin and compared structural properties of reduced and oxidized forms of the protein in solution by Raman spectroscopy. Ionization and hydrogen-bonding states of the two cysteine sulfhydryls (Cys32 and Cys35) in reduced thioredoxin were determined by monitoring the complex Raman SH stretching band (Li & Thomas, 1991) as a function of pH in the range 4.0 < pH < 12.2. The Raman SH markers indicate the following: (i) Both sulfhydryls of the native protein are relatively robust hydrogen-bond donors, but one is a stronger hydrogen-bond donor than the other. (ii) The sulfhydryl which donates the weaker hydrogen bond, assigned tentatively to Cys32, is preferentially titrated to the thiolate ion (S-) as the solution pH is increased from 4 to 7. Water or a hydroxyl oxygen is the probable acceptor for the Cys32 S-H donor. (iii) The sulfhydryl which donates the stronger hydrogen bond, Cys35, resists substantial ionization until roughly 50% of the more acidic sulfhydryl has been titrated. A carbonyl oxygen is proposed as the likely acceptor of the Cys35 S-H donor. (iv) The Raman titration data indicate pK1 = 7.1 +/- 0.2 and pK2 = 7.9 +/- 0.2 for the two thiol-thiolate equilibria. The lower pKa, which is the more strongly perturbed, is assigned tentatively to Cys32.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

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.

Base Sequence↗

PubMed1993

Structural studies of the enveloped dsRNA bacteriophage phi 6 of Pseudomonas syringae by Raman spectroscopy. I. The virion and its membrane envelope.

We report and interpret the first Raman spectrum of a double-stranded RNA virus containing a membrane envelope. Spectra of the native bacteriophage phi 6 and of its isolated host-attachment (spike) protein and phospholipid-free core assembly were collected from aqueous solutions over a wide range of temperature. Comparison of the vibrational spectra by digital difference methods permits the following structural conclusions regarding molecular constituents of the fully assembled virion. (1) The double-stranded RNA, phospholipid and protein components of the phage exhibit Raman amplitudes in accordance with their biochemically determined compositions in the native virion (10, 20 and 70%, respectively). (2) alpha-Helix and irregular conformations are the dominant secondary structures in proteins of both the viral membrane and nucleocapsid. This represents a departure from previously examined icosahedral phage and plant viruses, which are dominated by beta-sheet structures. (3) The phospholipids of the viral membrane are liquid crystalline throughout the determined range of virus thermostability (0 to 40 degrees C). (4) The P3 spike protein of phi 6, which is anchored to, but not sequestered within the viral membrane, is largely alpha-helical (approximately 35%) and highly thermolabile. Denaturation of P3 at temperatures above 30 degrees C leads to appreciable loss (approximately 20%) of alpha-helix in favor of beta-strand structure, and alters significantly the environments of many aromatic side-chains. (5) The secondary structures of integral membrane proteins of phi 6 are overwhelmingly alpha-helical (approximately 70 to 80%) and also thermolabile. In contrast to P3, which exhibits aspartate and glutamate carboxyls in the ionized form (CO2-), the integral membrane proteins exhibit only protonated carboxyl groups (COOH). Treatment of phi 6 with butylated hydroxytoluene (BHT), which has been shown to remove the P3 spike protein, does not significantly perturb phospholipids and associated integral proteins of the viral membrane or structural proteins and packaged double-stranded RNA of the nucleocapsid. However, P3 subunits, which are recovered after BHT treatment, exhibit radically altered secondary and tertiary structures, including the loss of most subunit alpha-helices. Among the P3 side-chains affected by BHT treatment, we note a general trend toward greater hydrophilicity and greater solvent exposure of the aromatic residues Trp and Tyr. On the other hand, the cysteine sulfhydryl groups of the BHT-isolated P3 monomer are not solvent exposed and function as strong hydrogen-bond donors in the protein core.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage phi 6↗

PubMed1993

Structural studies of the enveloped dsRNA bacteriophage phi 6 of Pseudomonas syringae by Raman spectroscopy. II. Nucleocapsid structure and thermostability of the virion, nucleocapsid and polymerase complex.

Structures and thermostabilities of the double-stranded (ds) RNA bacteriophage phi 6 and of its isolated nucleocapsid-polymerase complex (nucleocapsid core) and dsRNA components have been investigated by Raman spectroscopy. The spectra show that proteins of the phi 6 virion are collectively deficient in beta-sheet secondary structure. In particular, the major protein (P8) of the outer spherical shell of the phi 6 nucleocapsid exhibits a secondary structure dominated largely by alpha-helix and irregular conformations. The absence of appreciable beta-structure in the P8 subunit suggests a tertiary conformation lacking the beta-barrel motif common to subunits of most other spherical viral capsids. In addition, the Raman spectra show that subunits of the dodecahedral nucleocapsid core are also predominantly alpha-helical. The results thus indicate a largely alpha-helical secondary structure for the major subunit (P1) of the phi 6 nucleocapsid core, as well as for the P8 subunit of the outer spherical shell. Using Raman difference spectroscopy, we demonstrate that proteins of the nucleocapsid core (P1, P2, P4 and P7) interact extensively with the packaged phi 6 RNA genome, and further, that conformational stability of the packaged RNA is reduced upon removal from the core. Also, we find that proteins of the phi 6 nucleocapsid are significantly more thermostable than proteins of the viral membrane envelope, which are reported in the accompanying paper (Li et al., 1993). The present results suggest that both the architectural principles and modes of protein-RNA interaction in the phi 6 virion differ fundamentally from those of icosahedral single-stranded RNA viruses. Both Raman and circular dichroism spectra indicate that the dsRNA genome of phi 6 is an A-form structure. The Raman marker bands signify the presence only of C3'-endo/anti nucleoside conformers. The Raman signature of dsRNA, revealed in the spectrum of the phi 6 genome, is discussed here as a model for assessing base-pairing and base-stacking interactions in other ribonucleoprotein assemblies.

Bacteriophage phi 6↗

PubMed1993

Subunit conformational changes accompanying bacteriophage P22 capsid maturation.

In double-stranded DNA bacteriophages, packaging of dsDNA requires the transformation of a precursor procapsid into a mature viral capsid. Lattice expansion and release of scaffolding subunits accompanying DNA packaging. Three-dimensional structures of procapsid and mature phage lattices demonstrate that the capsid transformation involves substantial changes in subunit environment. Since this transformation occurs without subunit dissociation, it represents a transition between at least two stable subunit conformations. Using Raman spectroscopy, we have identified changes in coat protein secondary structure and side-chain environments which accompany the capsid transformation. The subunits of procapsid shells contain only 2.0 +/- 0.4% more alpha-helix and less beta-sheet than those of mature capsids; however, numerous side chains are substantially altered by the transformation, including tyrosines, tryptophans, phenylalanines, and aliphatics, which are widely distributed through the subunit sequence. We propose, therefore, that procapsid expansion is accomplished through the relative motion of coat subunit domains with little change in secondary structure. Such hinge-bending conformational transitions may couple ATP-dependent dsDNA condensation with shell expansion.

Bacteriophage P22↗

PubMed1993

Cysteine conformation and sulfhydryl interactions in proteins and viruses. 3. Quantitative measurement of the Raman S-H band intensity and frequency.

The bond stretching vibration of the cysteine sulfhydryl (SH) group in a typical protein generates a Raman band in the spectral interval 2500-2600 cm-1, a region devoid of interference from any other fundamental mode of vibration of the protein. The relatively high Raman cross section associated with the S-H stretching vibration, the sensitivity of the vibrational frequency to hydrogen bonding interactions and side chain configurations, and the dependence of the Raman intensity on thiol-thiolate equilibria, combine to make the Raman SH band a potentially valuable marker of protein sulfhydryl interactions and a unique indicator of sulfhydryl participation in thiol-disulfide oxidoreductase activity. In order to exploit Raman spectroscopy for these purposes, accurate and precise measurements of Raman SH band profiles are required. We show here that the required precision and accuracy can be achieved by use of the Raman band corresponding to the stretching vibration of in situ nitrogen gas as a quantitative intensity and frequency standard. The Raman Q-branch center of the N2 band occurs at 2330.7 cm-1.

Bacteriophage P22↗

PubMed1993

Raman spectroscopy of DNA-metal complexes. I. Interactions and conformational effects of the divalent cations: Mg, Ca, Sr, Ba, Mn, Co, Ni, Cu, Pd, and Cd.

Interactions of divalent metal cations (Mg2+, Ca2+, Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) with DNA have been investigated by laser Raman spectroscopy. Both genomic calf-thymus DNA (> 23 kilobase pairs) and mononucleosomal fragments (160 base pairs) were employed as targets of metal interaction in solutions containing 5 weight-% DNA and metal:phosphate molar ratios of 0.6:1. Raman difference spectra reveal that transition metal cations (Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) induce the greatest structural changes in B-DNA. The Raman (vibrational) band differences are extensive and indicate partial disordering of the B-form backbone, reduction in base stacking, reduction in base pairing, and specific metal interaction with acceptor sites on the purine (N7) and pyrimidine (N3) rings. Many of the observed spectral changes parallel those accompanying thermal denaturation of B-DNA and suggest that the metals link the bases of denatured DNA. While exocyclic carbonyls of dT, dG, and dC may stabilize metal ligation, correlation plots show that perturbations of the carbonyls are mainly a consequence of metal-induced denaturation of the double helix. Transition metal interactions with the DNA phosphates are weak in comparison to interactions with the bases, except in the case of Cu2+, which strongly perturbs both base and phosphate group vibrations. On the other hand, the Raman signature of B-DNA is largely unperturbed by Mg2+, Ca2+, Sr2+, and Ba2+, suggesting much weaker interactions of the alkaline earth metals with both base and phosphate sites. A notable exception is a moderate perturbation by alkaline earths of purine N7 sites in 160-base pair DNA, with Ca2+ causing the greatest effect. Correlation plots demonstrate a strong interrelationship between perturbations of Raman bands assigned to ring vibrations of the bases and those of bands assigned to exocyclic carbonyls and backbone phosphodiester groups. However, strong correlations do not occur between the Raman phosphodioxy band (centered near 1092 cm-1) and other Raman bands, suggesting that the former is not highly sensitive to the structural changes induced by divalent metal cations. The structural perturbations induced by divalent cations are much greater for > 23-kilobase pair DNA than for 160-base pair DNA, as evidenced by both the Raman difference spectra and the tendency toward the formation of insoluble aggregates. In the presence of transition metals, aggregation of high-molecular-weight DNA is evident at temperatures as low as 11 degrees C. A relationship between DNA melting and aggregation is proposed in which initial metal binding at major groove sites locally destabilizes the B-DNA double helix, causing displacement of the bases away from one another and exposing additional metal binding sites. Metal cation linkage of two displaced bases would allow separate DNA strands to crosslink. Aggregation is proposed to result from the formation of an extended network of these crosslinks.

Animals↗

PubMed1993

Raman dynamic probe of hydrogen exchange in bean pod mottle virus: base-specific retardation of exchange in packaged ssRNA.

We describe a novel approach to investigating exchange kinetics in biological assemblies. The method makes use of a Raman multichannel analyzer coupled with a dialysis flow cell. We employ this methodology to determine exchange rates of labile hydrogens in both the packaged RNA genome and protein subunits of bean pod mottle virus (BPMV). In the BPMV assembly, which is similar to human picornaviruses, the x-ray structure indicates that about 20% of the ssRNA chain is ordered at the threefold vertices of the icosahedral capsid, although the nucleotide bases in the ordered segments are not known (Chen et al., 1989). Here, we compare exchange profiles of the native virus with those of the empty capsid, model nucleic acids and aqueous solvent to reveal the following exchange characteristics of BPMV RNA and protein: (i) Base-specific retardation of exchange is observed in the packaged RNA. (ii) Retardation is greatest for uracil residues, for which the first-order exchange rate constant (kU = 0.18 +/- 0.02 min-1) is 40% lower than that of either the H2O solvent or adenine or cytosine groups of RNA (ksolv approximately kA approximately kC = 0.30 +/- 0.02 min-1). (iii) Retardation of exchange is also observed for the guanine residues of packaged RNA. (iv) No appreciable exchange of amide NH groups of capsid subunits occurs within the time of complete exchange (t approximately 10 min) of packaged RNA or bulk solvent. Thus, the present results identify sites in both the protein subunits (amide NH) and RNA nucleotides (amino NH2 and imino NH) which are resistant to solvent-catalyzed hydrogen exchange. We propose that retardation of exchange of labile sites of the RNA nucleotides is a consequence of the organization of the RNA chromosome within the virion. Our findings support a model for BPMV in which surface and buried domains of capsid subunits are extensively and rigidly hydrogen-bonded, and in which uracil and guanine exocyclic donor groups of packaged RNA are the principal targets for subunit interaction at the threefold vertices of the capsid.

Biophysical Phenomena↗

PubMed1993

Transmitter amino acid neurochemistry in chronic alcoholism with and without cirrhosis of the liver.

Neurotransmitter receptor studies are beginning to be applied more widely to human brain tissue obtained at autopsy. By taking tissue from well-documented cases which have been extensively characterized on histological and morphometric criteria, it is becoming possible to make clinicopathological correlations in studies of the effects of chronic alcohol abuse. Recent findings of alterations in the nature and amounts of amino acid neurotransmitter receptors in alcoholism are summarized, with special emphasis on the effects of chronic severe liver disease. There are selective changes in receptors in the superior frontal cortex of alcoholics. There is a marked increase in the density of the GABA binding site, and a lesser change in the density of the 'central-type' benzodiazepine site, on the GABAA-benzodiazepine receptor complex. In contrast, glutamate receptors may be much less affected. Together with morphological and cognitive studies, the results suggest that the superior frontal cortex is preferentially damaged in chronic alcoholism. An increase in 'central-type' benzodiazepine sites in both superior frontal cortex and motor cortex in cirrhotic alcoholics may reflect a more global brain damage, as observed in morphological studies. However, it should be noted the changes in [3H]GABA/muscimol binding were less pronounced in cirrhotic alcoholics than in non-cirrhotic alcoholics.

Journal Article↗

PubMed1992

Secondary structure and interactions of the packaged dsDNA genome of bacteriophage P22 investigated by Raman difference spectroscopy.

Vibrational spectra of the double-stranded DNA genome of bacteriophage P22 in packaged and unpackaged states are compared by digital difference Raman spectroscopy. The difference Raman spectrum, which is sensitive to structural changes at the level of < 2% of a given nucleotide type, reveals the effects of packaging upon sugar pucker, glycosyl orientation, phosphodiester geometry, base pairing, base stacking, and the electrostatic environment of DNA phosphate groups. For both packaged and unpackaged states, the experiments were performed on aqueous solutions at 25 degrees C containing effective P22 DNA concentrations of 30-50 mg/mL in 200 mM NaCl + 10 mM MgCl2 + 10 mM Tris at pH 7.5. At the experimental conditions employed, the B-form secondary structure of unpackaged P22 DNA is minimally perturbed by packaging the viral genome in the virion capsid. However, the electrostatic environment of DNA phosphates is dramatically altered with packaging. Specifically, we find the following: (1) C2'-endo sugar pucker and anti glycosyl orientations are conserved for all nucleosides. (2) Watson-Crick base pairing is essentially completely retained. (3) Alternative secondary structures, whether right- (A or C form) or left-handed (Z form), are not evident in either the packaged or unpackaged viral genome. (4) Small Raman hyperchromic effects (< 10%) observed for certain marker bands of dG, dA, and dT in the packaged state of P22 DNA suggest slightly reduced base-stacking interactions with packaging. These are consistent with previously reported UV hyperchromic effects, but the Raman spectrum shows that they are not associated with either base unpairing or strand separation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophages↗

PubMed1992

Conformations, interactions, and thermostabilities of RNA and proteins in bean pod mottle virus: investigation of solution and crystal structures by laser Raman spectroscopy.

We report and interpret laser Raman spectra of the three virion components of bean pod mottle virus (BPMV). The top component of BPMV is an empty capsid; middle and bottom components package the RNA2 and RNA1 genome segments, respectively. All components were investigated as both single crystals and aqueous solutions, the latter over wide ranges of temperature and ionic strength. The isolated RNA2 molecule of BPMV middle component was also investigated in both H2O and D2O solutions. The results permit assessment of RNA and protein structures, their mutual interactions in the virions, and their conformational thermostabilities and comparison of these structural characteristics for solution and crystal states of the particles. The principal findings of this study are (i) The extent of ordered A-form backbone (74%) and of base pairing (38% AU + 22% GC) in unpackaged (aqueous) RNA2 are significantly altered by packaging. The A-form secondary structure of RNA2 is increased by 12 +/- 4%, and guanine base interactions are also substantially increased with packaging. (ii) The thermostability of Raman-monitored secondary structure of unpackaged RNA2 (Tm approximately 43 degrees C) is greatly increased in the packaged state (Tm approximately 53 degrees C). This increase corresponds to a stabilization of the A-form backbone geometry in 15 +/- 5% of genome nucleotides. (iii) Packaging of RNA2 in the middle component stabilizes subunit-subunit interactions of the capsid, as evidenced by a thermal denaturation temperature Td approximately 65 degrees C for the virion, compared with Td approximately 55 degrees C for the empty capsid. (iv) Raman marker-band shifts implicate the purine 7N sites of RNA2 and aromatic side chains of subunits as the principal targets for RNA-subunit interaction. (v) At the conditions of the present experiments (8 degrees C, pH approximately 7, moderate ionic strength), the subunit secondary structures observed for solutions of the top, middle, and bottom components are indistinguishable by Raman spectroscopy from secondary structures observed for corresponding crystalline samples. (vi) On the other hand, side chains of subunits in the top component (empty capsid) yield significantly different Raman intensities in crystalline and solution states. These differences are interpreted as the result of changes in a small number of side-chain environments between crystal and solution. (vii) Similarly, small differences exist between RNA Raman markers of crystalline and aqueous virions, which are attributed to altered environments of nucleotide residues and to a small increase in the amount of A-form backbone geometry upon going from the crystal to the solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗

PubMed1992

Deuterium exchange of operator 8CH groups as a Raman probe of repressor recognition: interactions of wild-type and mutant lambda repressors with operator OL1.

The rate of deuterium exchange of a purine 8CH group in DNA is highly sensitive to both macromolecular secondary structure and intermolecular interactions which restrict solvent access to the major groove [Lamba, O.P., Becka, R., & Thomas, G.J., Jr. (1990) Biopolymers 29, 1465-1477]. We have exploited the sensitivity of the 8CH----8CD reaction to probe DNA recognition by the helix-turn-helix (HTH) motif of phage lambda cI repressor. We find that purine exchanges in the 19-base-pair OL1 operator are strongly and specifically restricted by binding of the HTH N-terminal domain of the repressor fragment (RF) comprising residues 1-102. The kinetics indicate large-scale obstruction of solvent access to operator 7N-8C purine sites. Interpretation of the exchange kinetics using a simple model suggests that only 7 purine residues (5 of 10 adenines and 2 of 9 guanines) remain unrestricted with respect to 8CH exchange in complexes of OL1 with the wild-type repressor. On the other hand, the 8CH exchange profile for the complex of OL1 with the Tyr88----Cys mutant repressor indicates that 9 purines (7 adenines and 2 guanines) are exchangeable. These results suggest important differences in major groove recognition in the two complexes. The proposed 8CH labeling profiles are consistent with molecular models of related complexes determined by X-ray crystallography [Jordan, S.R., & Pabo, C.O. (1988) Science 242, 893-899] and indicate that the structures observed in the crystal are largely maintained in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗

PubMed1992

Proteinases and glomerular matrix turnover.

The mesangial cell MMPs and their inhibitor may represent proteins through which biological modifiers such as cytokines and growth factors can control and influence the organization of the glomerulus. At the present we can only speculate on their exact function in glomerular disease. They could conceivably play a role in glomerular conditions where mesangial hypercellularity and cytoplasmic interposition between the endothelium and the basement membrane are frequent occurrences, and in which cytokine-enhanced synthesis of matrix-degrading enzymes could result in severe structural damage. A number of glomerular diseases such as diabetic nephropathy are characterized by the accumulation of glomerular matrix proteins. This could be explained by the inappropriate expression of intrinsic mesangial cell MMPs or TIMP. Further investigation of these proteins in experimental models of glomerular disease or in situ hybridization studies using the available probes promises to be a rewarding area of research during the next few years. For these studies the availability of rTIMP and recent developments in the design of synthetic inhibitors of MMPs may allow more searching investigations into the role of MMP in the pathophysiology of glomerular disease.

Amino Acid Sequence↗

PubMed1992

Amino acid neurotransmitter receptor changes in cerebral cortex in alcoholism: effect of cirrhosis of the liver.

Gamma-aminobutyric acidA/benzodiazepine receptor binding sites and the N-methyl-D-aspartate subclass of glutamate receptor sites were assessed in synaptic plasma membrane homogenates of cerebral cortex tissue obtained at autopsy from cirrhotic and noncirrhotic alcoholic patients and matched control subjects. The alcoholic patients consumed an average of greater than 80 g of ethanol/day, the control subjects less than 20 g/day. Postmortem delays up to approximately 100 h caused no significant loss of any of the binding sites; the patient and subject groups were closely matched for age. The affinities (KD) of the receptor sites did not differ between the patient and subject groups, nor between cortical regions. Using three different radioligands ([3H]muscimol, [3H]flunitrazepam, and [3H]diazepam), the gamma-aminobutyric acidA/benzodiazepine receptor complex was found to have greater density (Bmax) in superior frontal gyrus in alcoholic patients (which selectively shows morphological change in alcoholic patients), but was unchanged in motor cortex. Alcoholic patients with cirrhosis had much less pronounced changes. The density of the N-methyl-D-aspartate subclass of glutamate receptors, assessed with [3H]MK-801, did not vary across patient and subject groups.

Adult↗