Conformational analysis of thymidylate synthase from amino acid sequence and circular dichroism

Circular dichroism studies were carried out in the vacuum ultraviolet region for thymidylate synthase from Lactobacillus casei and its ligand complexes. The CD spectrum was analyzed for secondary structure by our method and the variable selection method, and both gave similar results. Our method predicts 33% alpha-helix, 25% (23% antiparallel and 2% parallel) beta-sheet, 20% turns, and 16% other structure. The secondary structure of this protein was also predicted from the amino acid sequence by four different methods. Though there is a variation in the prediction among these methods, the prediction of 32% alpha-helix and 23% beta-sheet by combining the four methods is in excellent agreement with our CD results. Further, the location of the predicted regions of alpha-helices and beta-strands along the sequence and the CD characteristics strongly suggest that this protein belongs to an alpha + beta structural class. Binding of the inhibitor FdUMP or the cofactor 5,10-methylenetetrahydrofolate did not change the CD spectrum. However, when both ligands were present, there was a significant change in the CD spectrum and the maximum changes occurred when the concentration of FdUMP was 1 mol/mol of enzyme. The addition of FdUMP and cofactor causes, respectively, a 5% and 6% decrease in beta-sheet and beta-turns and about an 8% increase in "other" structure.     

Conformational analysis of a snake neurotoxin by prediction from sequence, circular dichroism, and raman spectroscopy.

The secondary structure of the major neurotoxin from the sea snake Lapemis hardwickii was investigated by several methods of conformational analysis: structure prediction, circular dichroism, and laser Raman spectroscopy. From the primary structure, secondary structure prediction yielded two regions of β-sheet structure at residues 1–7 and 41–45. β-Turns were predicted at residues 14–17, 20–23, 30–33, 37–40, and 46–49. From the predictions, the toxin appears to be composed of approximately 20% β-sheet and 33% β-turn. The CD spectrum of the native toxin appears to be a hybrid of model spectra for β-sheet and β-turn proteins. The pH perturbation studies on the toxin observed by CD demonstrated that the toxin is a very stable molecule except at extremely high or low pH values. The Raman data indicated that the toxin contains both antiparallel β-sheet and β-turn structure. Using two methods of secondary structure quantitation from Raman spectra the molecule was calculated to contain 35% β-sheet from one method and 27% from the other. Overall, the various methods demonstrate that the toxin is composed of β-sheet and β-turn structure with little or no α-helix present. From the comparison of these different techniques appreciation can be gained for the necessity of several methods when identifying and quantitating secondary structure.     

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.     

Conformational analysis of hemopexin by fourier-transform infrared and circular dichroism spectroscopy

Hemopexin is a serum glyco-protein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptormediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% β-structure, 15% α-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme–hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the hemeprotein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis. © 1994 Wiley-Liss, Inc.     

A new small myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis)

Fast atom bombardment (FAB) mass spectrometry was used to identify a new small myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis). FAB mass spectrometry and Edman degradation were used to characterize its structure. This toxin is similar to myotoxin I from C. v. concolor, except that it possesses an additional. C-terminal asparaginyl-alanine. At 45 residues it is the longest known myotoxin a homolog. A myotoxin of 43 residues, identical to myotoxin I from C. v. concolor, was also found. To date no other species has been shown to produce more than one length of myotoxin. The present paper documents 42-, 43-, and 45-residue myotoxins from the venom of a single animal.     

The purification and amino acid sequence of toxin CM-13b from Naja haje annulifera (Egyptian cobra) venom.

Toxin CM-13b was purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. The toxin comprises 65 amino acid residues and is cross-linked by five disulphide bridges. The complete amino acid sequence of toxin CM-13b was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides purified by DEAE-cellulose chromatography and chromatography or electrophoresis on paper. The amino acid sequences of the intact toxin and its constituent peptides were determined by the Edman-Begg procedure, either through the use of the automatic sequenator or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The primary structure of toxin CM-13b shows a high degree of homology with that of protein S4C11 from Naja melanoleuca venom[1], but their toxicities are very different.     

Isolation, stabilization, and characterization of a toxin from timber rattlesnake venom.

A rapid and convenient method for the purification of a toxin from timber rattlesnake, Crotalus horridus horridus, venom using carboxymethyl cellulose ion-exchange chromatography has been devised. The toxicity of this venom component is labile, but it is stabilized by the addition of 20+ V/V glycerol to the buffer solution. This toxin has a molecular weight of 15,000 +/- 700 as determined by SDS gel electrophoresis. It is both heat and protease resistant. Treatment of this venom component with 2-mercaptoethanol followed by G-50 Sephadex chromatography causes no loss of toxicity although incubation of the toxin with 1% SDS and 1% 2-mercaptoethanol prior to electrophoresis does result in a faster migrating species. The toxin does not affect neuromuscular junctions but does appear to act on the nervous system. It causes no local responses in mice.     

Amino acid sequence and circular dichroism of Indian cobra (Naja naja naja) venom acidic phospholipase A2

The full amino acid sequence of the acidic phospholipase A2 from Indian cobra (Naja naja naja) venom was determined and its tertiary structure examined by circular dichroism (CD). The sequence was aligned with other sequences of secreted phospholipase A2 from snakes of the genus Naja, using the progressive alignment method of Feng and Doolittle (J. Mol. Evol. (1987) 25, 351-360). The primary sequence of Naja naja naja phospholipases A2 shows up to 85% identity with the other acidic Naja phospholipase A2. CD studies indicate a 40-50% alpha-helical content in a tertiary structure which resists denaturation at high temperature, with or without chaotropic salts.     

Conformational analysis of apolipoprotein A-I and E-3 based on primary sequence and circular dichroism.

The primary and secondary structure of human plasma apolipoprotein A-I and apolipoprotein E-3 have been analyzed to further our understanding of the secondary and tertiary conformation of these proteins and the structure and function of plasma lipoprotein particles. The methods used to analyze the primary sequence of these proteins used computer programs: (a) to identify repeated patterns within these proteins on the basis of conservative substitutions and similarities within the physicochemical properties of each residue; (b) for local averaging, hydrophobic moment, and Fourier analysis of the physicochemical properties; and (c) for secondary structure prediction of each protein carried out using homology, statistical, and information theory based methods. Circular dichroism was used to study purified lipid-protein complexes of each protein and quantitate the secondary structure in a lipid environment. The data from these analyses were integrated into a single secondary structure prediction to derive a model of each protein. The sequence homology within apolipoproteins A-I, E-3, and A-IV is used to derive a consensus sequence for two 11 amino acid repeating sequences in this family of proteins.     

Structural properties of mojave toxin of crotalus scutulatus (Mojave rattlesnake) determined by laser Raman spectroscopy.

Laser Raman Spectra were obtained on aqueous and solid samples of Mojave toxin isolated from the venom of the Mojave rattlesnake ($\text{Crotalus}\text{scutulatus}$). The Raman spectra reveal that the Mojave toxin, an acidic protein of molecular weight about 22,000, contains a predominantly α-helical secondary structure and that the tyrosyl residues, on the basis of the Raman frequencies and intensities, are exposed to the solvent. These features of the Mojave toxin distinguish it structurally from the neurotoxins of sea snake venoms. However, like the sea snake venom toxins, Mojave toxin contains four disulfide bridges and is not greatly altered in structure by removal of the aqueous solvent.     

The amino acid sequence of toxin V II 2, a cytotoxin homologue from banded Egyptian cobra (Naja haje annulifera) venom.

Toxin V II 2 comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The complete amino acid sequence of this toxin was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides were purified by ion-exchange chromatography and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequenator or by manual manipulation, was employed to obtain the sequence of the intact toxin and the pure peptides. The chymotryptic digest provided the necessary overlapping peptides which allowed the alignment of tryptic peptides. The amino acid sequence of Naja haje annulifera toxin V II 2 shows a high degree of homology with cytotoxin V II 1 of the same venom.     

Biochemical characterization of hemorrhagic toxin from crotalus viridis viridis (prairie rattlesnake) venom

Hemorrhage, necrosis and edema are some of the effects often observed following snake bites. This paper reports studies on the isolation and biological properties of hemorrhagic toxin from Crotalus viridis viridis (Prairie rattlesnake) venom. A hemorrhagic toxin was isolated from C. v. viridis venom by Sephadex G-50, DEAE-Sephacel and Q-Sepharose column chromatographies.The hemorrhagic toxin from C. v. viridis venom was shown to be homogenous as demonstrated by a single band on polyacrylamide gel electrophoresis and immunodiffusion. Its molecular weight was approximately 54,000 dallons, and it contained 471 amino acid residues. The toxin possessed hemorrhagic activity with a minimum hemorrhagic dose (MHD) of 0.11 μ g, and hydrolytic activity on dimethylcasein, casein, azocasein, azoalbumin, azocoll and hide powder azure. Hemorrhagic and casein hydrolytic activities were inhibited by EDTA, o-phenanthroline or dithiothreitol. The toxin contained 1 mole of zinc per mole of protein and zinc is essential for both hemorrhagic and proteolytic activities. Hemorrhagic toxin possessed hydrolytic activity on the B-chain of insulin, which cleaves His(5)-Leu(6), His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25) bonds. This toxin also hydrolyzed Aα and Bβ chains of fibrinogen. Intramuscular injections of hemorrhagic toxin caused an increase of creatine phosphokinase activity in mice serum from 50.3 mU/ml to 1133 mU/ml. A toxin isolated from C. v. viridis venom was shown to have strong hemorrhagic activity. Partial characterization is reported for this major hemorrhagic toxin in C. v. viridis venom.     

Characterization of two hemorrhagic zinc proteinases, toxin c and toxin d, from western diamondback rattlesnake (Crotalus atrox) venom

Two hemorrhagic proteinases from Crotalus atrox venom, hemorrhagic toxin c (Ht-c) and hemorrhagic toxin d (Ht-d), were characterized and compared to one another. The two toxins are zinc metalloproteinases which both have molecular weights of 24,000. Their isoelectric points are slightly acidic, Ht-c being the more basic of the two with an isoelectric point of 6.2, whereas Ht-d has an isoelectric point of 6.1. Only minor differences were found in the amino acid compositions of the two toxins. The toxins were both demonstrated to be hemorrhagic, using an in vivo assay, and also proteolytic. Prior treatment of the hemorrhagic proteinases with ethylenediaminetetraacetic acid and o-phenanthroline eliminated both the hemorrhagic and the proteolytic activities. Aprotinin and phenylmethylsulfonyl fluoride had no effect upon these activities. The pH optimum of the proteolysis by Ht-c and Ht-d on hide powder azure as the substrate was between pH 8 and pH 9. The circular dichroism spectra for Ht-c and Ht-d appear almost identical with respect to minima positions and elipticities, indicative of very similar solution structures for the two enzymes. Antiserum raised in mice against Ht-c was assayed on double-diffusion Ouchterlony plates for cross-reactivity with other hemorrhagic toxins from C. atrox venom. From this experiment it was concluded that the two hemorrhagic proteinases Ht-c and Ht-d share identical antigenic structures. This was corroborated by tryptic mapping of the two toxins. Only one major difference was observed from the maps. In the case of Ht-c, it was determined that an aspartate was substituted by an alanine when compared to Ht-d. From these characterization studies we conclude that Ht-c and Ht-d are isoenzymes with only very minor differences in their structures.