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 protein's 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.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Deep-level diagnostic value of the rDNA-ITS region

The similarity of certain reported angiosperm rDNA internal transcribed spacer (ITS) region sequences to those of green algae prompted our analysis of the deep-level phylogenetic signal in the highly conserved but short 5.8S and hypervariable ITS2 sequences. We found that 5.8S sequences yield phylogenetic trees similar to but less well supported than those generated by a ca. 10-fold longer alignment from rDNA-18S sequences, as well as independent evidence. We attribute this result to our finding that, compared to 18S, the 5.8S has a higher proportion of sites subject to vary and greater among-site substitution rate homogeneity. We also determined that our phylogenetic results are not likely affected by intramolecular compensatory mutation to maintain RNA secondary structure nor by evident systematic biases in base composition. Despite historical homology, there appears to be no ITS2 primary sequence similarity shared sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that groups, however, share sufficient similarity to cluster correctly on the basis of alignability. Our results indicate that ITS region sequences can diagnose organismal origins and phylogenetic relationships at many phylogenetic levels and provide a useful paradigm for molecular evolutionary study.      

Production of Serine Proteases by the Oyster Pathogen Perkinsus marinus (Apicomplexa) In Vitro

ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.     

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.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.