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Biomedical subjects

F Jurnak

Publications and source records attributed to F Jurnak.

At least 19 recordsLinked to original sources
PubMed1996

Differential effect of site-directed mutations in pelC on pectate lyase activity, plant tissue maceration, and elicitor activity.

Oligonucleotide site-directed mutations were introduced into the pelC gene of Erwinia chrysanthemi EC16 that directed single or double amino acid changes affecting disulfide linkages, calcium binding, catalysis, and protein folding. Subsequent characterization of the purified PelC mutant proteins demonstrated that pectinolytic function involves amino acids located near the calcium binding site rather than those surrounding an invariant vWiDH sequence. Wild-type PelC and the tested mutant proteins generally macerated plant tissue in proportion to their specific pectinolytic activity in vitro. However, some mutants gave higher maceration activity in plant tissue and elicited greater production of the phytoalexin, glyceollin, in soybean cotyledons than predicted by their in vitro pectinolytic activity. Most notable in this regard were three different mutations at lysine 172 with greatly reduced pectinolytic activity but as much elicitor activity as the wild-type protein. PelE macerated plant tissue 10 times more efficiently than PelC, as observed previously, but surprisingly showed equal activity in the elicitor assay. The results indicate that factors other than pectinolytic activity per se are involved in plant tissue maceration and elicitor activity.

Dickeya chrysanthemi↗
PubMed1996

An alpha to beta conformational switch in EF-Tu.

BACKGROUND: The bacterial elongation factor EF-Tu recognizes and transports aminoacyl-tRNAs to mRNA-programmed ribosomes. EF-Tu shares many structural and functional properties with other GTPases whose conformations are regulated by guanine nucleotides. RESULTS: An intact form of Escherichia coli EF-Tu complexed with GDP has been crystallized in the presence of the EF-Tu-specific antibiotic GE2270 A. The three-dimensional structure has been solved by X-ray diffraction analysis and refined to a final crystallographic R factor of 17.2% at a resolution of 2.5 A. The location of the GE2270 A antibiotic-binding site could not be identified. CONCLUSIONS: The structure of EF-Tu-GDP is nearly identical to that of a trypsin-modified form of EF-Tu-GDP, demonstrating conclusively that the protease treatment had not altered any essential structural features. The present structure represents the first view of an ordered Switch I region in EF-Tu-GDP and reveals similarities with two other GTPases complexed with GDP: Ran and ADP-ribosylation factor-1. A comparison of the Switch I regions of the GTP and GDP forms of EF-Tu also reveals that a segment, six amino acids in length, completely converts from an alpha helix in the GTP complex to beta secondary structure in the GDP form. The alpha to beta switch in EF-Tu may represent a prototypical activation mechanism for other protein families.

Anti-Bacterial Agents↗
PubMed1995

Circular dichroism of the parallel beta helical proteins pectate lyase C and E.

The pectate lyases, PelC and PelE, have an unusual folding motif, known as a parallel beta-helix, in which the polypeptide chain is coiled into a larger helix composed of three parallel beta-sheets connected by loops having variable lengths and conformations. Since the regular secondary structure consists almost entirely of parallel beta-sheets these proteins provide a unique opportunity to study the effect of parallel beta-helical structure on circular dichroism (CD). We report here the CD spectra of PelC and PelE in the presence and absence of Ca2+, derive the parallel beta-helical components of the spectra, and compare these results with previous CD studies of parallel beta-sheet structure. The shape and intensity of the parallel beta-sheet spectrum is distinctive and may be useful in identifying other proteins that contain the parallel beta-helical folding motif.

Bacterial Proteins↗
PubMed1995

Protein motifs. 3. The parallel beta helix and other coiled folds.

A new type of structural domain, composed of all parallel beta strands, has been observed within the last year. An analysis of the basic types suggests that there are two distinct classes: the parallel beta helices, which belong to a tri beta-strand category, and the beta roll, which belongs to a di beta-strand category. The novel structural features of each class are described and the proteins belonging to each category are summarized. Proteins with the parallel beta helix fold include three pectate lyases and the tailspike protein from P22 phage. Proteins with the beta roll fold include two alkaline proteases. Although the parallel beta composition is emphasized, the same set of proteins share another common structural feature with several other proteins containing alpha helices: the polypeptide backbone is folded into a coiled structure in which each coil has the same 3-dimensional arrangement of a group of secondary structural elements. In addition to parallel beta domains, the other groups include the alpha/beta coiled fold, as represented by ribonuclease inhibitor, and the alpha/alpha coiled fold, as represented by lipovitellin and soluble lytic transglycoslyase. Novel features of the alpha/beta and alpha/alpha coiled folds are summarized.

Crystallography, X-Ray↗
PubMed1995

Functional implications of structure-based sequence alignment of proteins in the extracellular pectate lyase superfamily.

Pectate lyases are plant virulence factors that degrade the pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular pectate lyases as well as all sequence homologs, including pectin lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel beta helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the pectate lyase structures, suggesting two distinct enzymatic functions for extracellular pectate lyases and their sequence homologs.

Amino Acid Sequence↗
PubMed1994

The ABC of EF-G.

The recently solved crystal structures of Thermus thermophilus elongation factor G, with and without GDP, reveal a protein of five domains with surprising features which can be correlated with biochemical data to suggest probable functional roles.

GTP Phosphohydrolase-Linked Elongation Factors↗
PubMed1994

Parallel beta-domains: a new fold in protein structures.

A new type of structural domain, composed of parallel beta-strands folded into a coiled structure, has been observed in several protein structures within the past year. An analysis of the basic motif indicates that there are two distinct types, with variations likely to be discovered in the future.

Amino Acid Sequence↗
PubMed1993

Unusual structural features in the parallel beta-helix in pectate lyases.

BACKGROUND: A new type of domain structure, an all parallel beta class, has recently been observed in two pectate lyases, PelC and PelE. The atomic models have been analyzed to determine whether the new tertiary fold exhibits unusual structural features. RESULTS: The polypeptide backbone exhibits no new types of secondary structural elements. However, novel features occur in the amino acid side chain interactions. The side chain atoms form linear stacks that include asparagine ladders, serine stacks, aliphatic stacks, and ringed-residue stacks. A new type of beta-sandwich between parallel beta-sheets is observed with properties that are more characteristic of antiparallel beta-sheets. CONCLUSION: An analysis of the PelC and PelE structures, belonging to an all parallel beta structural class, reveals novel amino acid side chain interactions, a new type of beta-sandwich and an atypical amino acid composition of parallel beta-sheets. The findings are relevant to three-dimensional structural predictions.

Amino Acid Sequence↗
PubMed1993

New domain motif: the structure of pectate lyase C, a secreted plant virulence factor.

Pectate lyases are secreted by pathogens and initiate soft-rot diseases in plants by cleaving polygalacturonate, a major component of the plant cell wall. The three-dimensional structure of pectate lyase C from Erwinia chrysanthemi has been solved and refined to a resolution of 2.2 angstroms. The enzyme folds into a unique motif of parallel beta strands coiled into a large helix. Within the core, the amino acids form linear stacks and include a novel asparagine ladder. The sequence similarities that pectate lyases share with pectin lyases, pollen and style proteins, and tubulins suggest that the parallel beta helix motif may occur in a broad spectrum of proteins.

Amino Acid Sequence↗
PubMed1991

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21.

We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange. We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems. The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding. On the other hand, no change in hydrogen bonding is observed at guanine's N7. The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond. These results are compared to previous structural and chemical studies.

Amino Acid Sequence↗
PubMed1991

Stabilization of the Escherichia coli elongation factor Tu-GTP-aminoacyl-tRNA complex.

The effect of ammonium sulfate on the Escherichia coli elongation factor Tu-GTP-aminoacyl-tRNA complex has been studied. The half-lives of 12 E. coli aminoacyl-tRNA species were determined at 37 degrees C in the presence and absence of an equimolar amount of EF-Tu-GTP and in the presence and absence of 1.5 M ammonium sulfate. The results indicate that the addition of 1.5 M ammonium sulfate to the ternary complex increased the stability of all 12 complexes studied. In addition, the effects of various salts and crystallization agents on the stability of the E. coli EF-Tu-GTP-phenylalanyl-tRNA complex was studied in detail. Binding parameters were also measured under various conditions at 37 degrees C. The results indicate that the stability and the Kassoc of the ternary complex, using phenylalanyl-tRNA, can be increased by the presence of polyethylene glycol or ammonium sulfate.

Escherichia coli↗
PubMed1990

Genetic engineering, isolation and characterization of a truncated Escherichia coli elongation factor Tu comprising domains 2 and 3.

A deletion mutant of a plasmid born Escherichia coli tufA gene, which codes for a truncated elongation factor Tu comprising domains 2 and 3, has been constructed by genetic engineering. This gene was overexpressed in E. coli, and a polypeptide representing the truncated elongation factor Tu was isolated, purified to near homogeneity, crystallized and characterized physico-chemically as well as biochemically. Circular dichroism spectroscopy and limited tryptic digestion demonstrate that the isolated domain pair 2 and 3 behaves like an independent folding unit which adopts a similar secondary and most likely, tertiary, structure to that present in the intact elongation factor Tu. However, the isolated domain pair 2 and 3 does not interact with aminoacyl-tRNA or the antibiotic kirromycin, two ligands which were shown previously by cross-linking experiments to be in contact with amino acid residues located in domains 1 and 2, and domain 3, respectively. The results suggest that the isolated domain pair 2 and 3 by itself forms too few contacts with these ligands to form a stable complex. Furthermore, the data suggest that domain 1 in intact EF-Tu, in a subtle but nevertheless decisive manner, alters the conformation of the other two domains in such a way that all three domains cooperatively create a high affinity binding site for aminoacyl-tRNA and the antibiotic kirromycin.

Circular Dichroism↗
PubMed1990

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu.

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.

Amino Acid Sequence↗
PubMed1990

Preliminary crystallographic analysis of the plant pathogenic factor, pectate lyase C from Erwinia chrysanthemi.

Pectate lyases are saccharide-binding enzymes that degrade plant cell walls. One pectate lyase from Erwinia chrysanthemi (EC16), termed pectate lyase C, has been crystallized from ammonium sulfate. The preliminary x-ray diffraction analysis indicates that the crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions, a = 73.4 A, b = 80.3 A, and c = 95.1 A. The crystals diffract to a resolution of 2.2 A and have one molecule/asymmetric unit.

Ammonium Sulfate↗
PubMed1990

Preliminary crystallographic analysis of a complex between tetracycline and the trypsin-modified form of Escherichia coli elongation factor Tu.

Crystals of a complex between the antibiotic tetracycline and the trypsin-modified form of the Escherichia coli protein elongation factor Tu have been grown in a form suitable for high-resolution X-ray diffraction analysis. The crystals belong to space group P2(1), with cell dimensions a = 69.7 A, b = 156.4 A, c = 135.4 A and beta = 95.3 degrees, and contain six molecules of the complex per asymmetric unit. The crystals are well ordered and diffract to a resolution of 2.3 A.

Escherichia coli↗