A fast method for predicting amino acid mutations that lead to unfolding.
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Amino acid mutation(s) that cause(s) partial or total unfolding of a protein can lead to disease states and failure to produce mutants. It is therefore very useful to be able to predict which mutations can retain the conformation of a wild-type protein and which mutations will lead to local or global unfolding of the protein. We have developed a fast and reasonably accurate method based on a backbone-dependent side-chain rotamer library to predict the (folded or unfolded) conformation of a protein upon mutation. This method has been tested on proteins whose wild-type 3D structures are known and whose mutant conformations have been experimentally characterized to be folded or unfolded. Furthermore, for the cases studied here, the predicted partially folded or denatured mutant conformation correlate with a decrease in the stability of the mutant relative to the wild-type protein. The key advantage of our method is that it is very fast and predicts locally or globally unfolded states fairly accurately. Hence, it may prove to be useful in designing site-directed mutagenesis, X-ray crystallography and drug design experiments as well as in free energy simulations by helping to ascertain whether a mutation will alter or retain the wild-type conformation. (+info)
Bicarbonate enhances peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical and scavenging of carbonate anion radical by metalloporphyrin antioxidant enzyme mimetics.
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Much evidence exists for the increased peroxidase activity of copper, zinc superoxide dismutase (SOD1) in oxidant-induced diseases. In this study, we measured the peroxidase activity of SOD1 by monitoring the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF). Bicarbonate dramatically enhanced DCFH oxidation to DCF in a SOD1/H(2)O(2)/DCFH system. Peroxidase activity could be measured at a lower H(2)O(2) concentration ( approximately 1 microm). We propose that DCFH oxidation to DCF is a sensitive index for measuring the peroxidase activity of SOD1 and familial amyotrophic lateral sclerosis SOD1 mutants and that the carbonate radical anion (CO(3)) is responsible for oxidation of DCFH to DCF in the SOD1/H(2)O(2)/bicarbonate system. Bicarbonate enhanced H(2)O(2)-dependent oxidation of DCFH to DCF by spinal cord extracts of transgenic mice expressing SOD1(G93A). The SOD1/H(2)O(2)/HCO(3)(-)-dependent oxidation was mimicked by photolysis of an inorganic cobalt carbonato complex that generates CO(3). Metalloporphyrin antioxidants that are usually considered as SOD1 mimetic or peroxynitrite dismutase effectively scavenged the CO(3) radical. Implications of this reaction as a plausible protective mechanism in inflammatory cellular damage induced by peroxynitrite are discussed. (+info)
A possible role for pi-stacking in the self-assembly of amyloid fibrils.
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Amyloid fibril formation is assumed to be the molecular basis for a variety of diseases of unrelated origin. Despite its fundamental clinical importance, the mechanism of amyloid formation is not fully understood. When we analyzed a variety of short functional fragments from unrelated amyloid-forming proteins, a remarkable occurrence of aromatic residues was observed. The finding of aromatic residues in diverse fragments raises the possibility that pi-pi interactions may play a significant role in the molecular recognition and self-assembly processes that lead to amyloid formation. This is in line with the well-known central role of pi-stacking interactions in self-assembly processes in the fields of chemistry and biochemistry. We speculate that the stacking interactions may provide energetic contribution as well as order and directionality in the self-assembly of amyloid structures. Experimental data regarding amyloid formation and inhibition by short peptide analogs also support our hypothesis. The pi-stacking hypothesis suggests a new approach to understanding the self-assembly mechanism that governs amyloid formation and indicates possible ways to control this process. (+info)
Exploring the active site of plant glutaredoxin by site-directed mutagenesis.
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Six mutants (Y26A, C27S, Y29F, Y29P, C30S and Y26W/Y29P) have been engineered in order to explore the active site of poplar glutaredoxin (Grx) (Y26CPYC30). The cysteinic mutants indicate that Cys 27 is the primary nucleophile. Phe is a good substitute for Tyr 29, but the Y29P mutant was inactive. The Y26A mutation caused a moderate loss of activity. The YCPPC and WCPPC mutations did not improve the reactivity of Grx with the chloroplastic NADP-malate dehydrogenase, a well known target of thioredoxins (Trxs). The results are discussed in relation with the known biochemical properties of Grx and Trx. (+info)
Charged amino acids conserved in the aromatic acid/H+ symporter family of permeases are required for 4-hydroxybenzoate transport by PcaK from Pseudomonas putida.
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Charged amino acids in the predicted transmembrane portion of PcaK, a permease from Pseudomonas putida that transports 4-hydroxybenzoate (4-HBA), were required for 4-HBA transport, and they were also required for P. putida to have a chemotactic response to 4-HBA. An essential amino acid motif (DGXD) containing aspartate residues is located in the first transmembrane segment of PcaK and is conserved in the aromatic acid/H+ symporter family of the major facilitator superfamily of transporters. (+info)
Aromatic-aromatic interactions in and around alpha-helices.
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To understand the role of aromatic-aromatic interactions in imparting specificity to the folding process, the geometries of four aromatic residues with different sequence spacing, located in alpha-helices or five residues from helical ends, interacting with each other have been elucidated. The geometry is found to depend on the sequence difference. Specific interactions (C-H...pi and N-H...pi) which result from this geometry may cause a given pair of residues (such as Phe-His) with a particular sequence difference to occur more than expected. The most conspicuous residue in an aromatic pair in the context of helix stability is His, which is found at the last (C1) position or the two positions (Ncap and Ccap) immediately flanking the helix. An alpha-helix and a contiguous 3(10)-helix or two helices separated by a non-helical residue can have interacting aromatic pairs, the geometry of interaction and the relative orientation between the helices being rather fixed. Short helices can also have interacting residues from either side. (+info)
The ARO4 gene of Candida albicans encodes a tyrosine-sensitive DAHP synthase: evolution, functional conservation and phenotype of Aro3p-, Aro4p-deficient mutants.
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The enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyses the first step in aromatic amino acid biosynthesis in prokaryotes, plants and fungi. Cells of Saccharomyces cerevisiae contain two catalytically redundant DAHP synthases, encoded by the genes ARO3 and ARO4, whose activities are feedback-inhibited by phenylalanine and tyrosine, respectively. ARO3/4 gene transcription is controlled by GCN4. The authors previously cloned an ARO3 gene orthologue from Candida albicans and found that: (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine, and (2) a homozygous aro3-deletion mutant of C. albicans is phenotypically Aro(+), suggesting the existence of another isozyme(s). They now report the identification and functional characterization of the C. albicans orthologue of S. cerevisiae Aro4p. The two Aro4p enzymes share 68% amino acid identity. Phylogenetic analysis places the fungal DAHP synthases in a cluster separate from prokaryotic orthologues and suggests that ARO3 and ARO4 arose from a single gene via a gene duplication event early in fungal evolution. C. albicans ARO4 mRNA is elevated upon amino acid starvation, consistent with the presence of three putative Gcn4p-responsive elements (GCREs) in the gene promoter sequence. C. albicans ARO4 complements an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess tyrosine. The authors engineered Deltaaro3/Deltaaro3 Deltaaro4/MET3p::ARO4 cells of C. albicans (with one wild-type copy of ARO4 placed under control of the repressible MET3 promoter) and found that they fail to grow in the absence of aromatic amino acids when ARO4 expression is repressed, and that this growth defect can be partially rescued by aromatic amino acids and certain aromatic amino acid pathway intermediates. It is concluded that, like S. cerevisiae, C. albicans contains two DAHP synthases required for the first step in the aromatic amino acid biosynthetic pathway. (+info)
Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase.
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The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase. (+info)