Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Mutant Proteins”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1986

Expression in Escherichia coli of synthetic human interleukin-1 alpha genes encoding the processed active protein, mutant proteins, and beta-galactosidase fusion proteins.

We have synthesized, cloned, and expressed the coding region for the C-terminal 159 amino acids (aa) of the human active interleukin polypeptide hormone IL-1 alpha. The sequence was assembled in stages and includes preferred Escherichia coli codons and unique restriction sites. The coding region was cloned on a multicopy plasmid vector adjacent to signals for transcription and translation that directed synthesis of 6% of total E. coli protein as IL-1 alpha. Active IL-1 alpha mutants that have a C-terminal additional eleven aa and that have N-terminal deletions of six and fourteen aa are described. Plasmids expressing beta-galactosidase fusion proteins with various parts of IL-1 alpha at their N-termini were constructed.

Base Sequence↗
PubMed1994

Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins.

Mutant (delta F508) and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) were synthesized initially as an approximately 140-kDa core-glycosylated precursor, which, in the case of wild-type CFTR, was chased to an approximately 160 kDa form bearing complex oligosaccharides. Mutant CFTR disappeared from the detergent-soluble cell extract with rapid (t1/2 = 27 min) kinetics. Only approximately 25% of the initially synthesized wild-type 140-kDa CFTR precursor was detected as mature protein; the remaining approximately 75% decayed with kinetics (t1/2 = 33 min) indistinguishable from those of the mutant. Rapid degradation kinetics and inefficient processing of wild-type CFTR were also observed in the colonic carcinoma lines HT29 and T84 and in stably transfected C127 cells, which express 5-50 times lower levels of CFTR. These results suggest that inefficient processing and rapid degradation of wild-type CFTR precursor are an intrinsic property of CFTR in these diverse cell types and are not an artifact of overexpression. Degradation of wild-type and mutant 140-kDa CFTR began without significant lag following synthesis. These data suggest that wild-type and delta F508 CFTR differ in the efficiency of folding of the core-glycosylated primary translation product.

Amino Acid Sequence↗
PubMed2006

Altered phosphorylation of cytoskeletal proteins in mutant protein phosphatase 2A transgenic mice.

Protein phosphatase 2A (PP2A) is a family of heterotrimeric enzymes with diverse functions under physiologic and pathologic conditions such as Alzheimer's disease. All PP2A holoenzymes have in common a catalytic subunit C and a structural scaffolding subunit A. These core subunits assemble with various regulatory B subunits to form heterotrimers with distinct functions in the cell. Substrate specificity of PP2A in vitro is determined by regulatory subunits with leucine 309 of the catalytic subunit C playing a crucial role in the recruitment of regulatory subunits into the complex. Here we expressed a mutant form of Calpha, L309A, in brain and Harderian (lacrimal) gland of transgenic mice. We found an altered recruitment of regulatory subunits into the complex, demonstrating a role for the carboxyterminal leucine of Calpha in regulating holoenzyme assembly in vivo. This was associated with an increased phosphorylation of tau in brain and an impaired dephosphorylation of vimentin demonstrating that both cytoskeletal proteins are in vivo substrates of distinct PP2A holoenzyme complexes.

Animals↗
PubMed1996

Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system.

A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central alpha-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.

Arginine↗
PubMed2001

Isolation of viral coat protein mutants with altered assembly and aggregation properties.

A method was developed to screen bacteria for synthesis of mutant proteins with altered assembly and solubility properties using bacteriophage MS2 coat protein as a model self-associating protein. Colonies expressing coat protein from a plasmid were covered with an agarose overlay under conditions that caused the lysis of some of the cells in each colony. The proteins thus liberated diffused through the overlay at rates depending on their molecular sizes. After transfer of the proteins to a nitrocellulose membrane, probing with coat protein-specific antiserum revealed spots whose sizes and intensities were related to the aggregation state of coat protein. The method was employed in the isolation of assembly defective mutants and to find soluble variants of an aggregation-prone coat protein mutant.

Amino Acid Substitution↗
PubMed1999

Experimental verification of the 'stability profile of mutant protein' (SPMP) data using mutant human lysozymes.

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishikawa,K., 1995, J. Mol. Biol., 248, 733-738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudo-energy potential developed for evaluating structure-sequence compatibility in the structure prediction method, the 3D-1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability of SPMP. The thermodynamic parameters for denaturation and crystal structures of these mutant proteins were determined. One mutant protein was stabilized as expected, compared with the wild-type protein. However, the others were not stabilized even though the structural changes were subtle, indicating that SPMP overestimates the increase in stability or underestimates negative effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP. The correlation of the stability changes between the experiment and prediction depended on the types of substitution: there were some correlations for proline mutants and cavity-creating mutants, but no correlation for mutants related to side-chain hydrogen bonds. The present results may indicate some additional factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.

Amino Acid Substitution↗
PubMed1993

Intestinal fatty acid binding protein: characterization of mutant proteins containing inserted cysteine residues.

Site-directed mutagenesis was used to introduce cysteine residues into the rat intestinal fatty acid binding protein, an almost all beta-sheet protein that in the wild-type contains neither cysteine nor proline residues. Six mutants (I23C, S53C, V60C, L72C, L89C, and A104C) with a single cysteine residue substituted for a hydrophobic residue were characterized by their stability toward denaturants at pH 7.2 and 9.6, by their fluorescent properties, and by their reactivity toward the sulfhydryl modifying reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 4,4'-dipyridyl disulfide (4-PDS). In terms of protein stability, the substitutions were reasonably conservative with only two (V60C and L89C) being somewhat less stable than the wild-type. The mutant proteins differed considerably, however, in their reactivity toward the modifying reagents. One residue, Cys89, located in a hydrophobic core near a turn between two beta-strands, was unreactive, while two residues, Cys60 and Cys104, located in the middle of beta-strands in the cavity into which fatty acid binds, reacted only very slowly and were further protected by oleate. Cys53, located near a turn and partially buried, appeared to have an unusually low pK value. Two residues, Cys23 and Cys72, reacted more rapidly in the native protein than in the unfolded protein. Both residues are located near the portal for the fatty acid binding, and one, Cys72, was strongly protected from modification by the presence of oleate. Examination of the crystal structure indicates that Cys72 is not easily solvent-accessible. We conclude that this high reactivity for this residue may be a consequence of rapid conformational flexibility in this region of the structure.

Animals↗
PubMed2002

Localization of lens intrinsic membrane protein MP19 and mutant protein MP19(To3) using fluorescent expression vectors.

PURPOSE: [corrected] MP19 is the second most abundant major intrinsic protein of the lens fiber cell membrane. A specific heritable mutation at amino acid 15 in the MP19 protein, termed MP19To3, results in total cataract and microphthalmia in the mouse. The goals of this study were to determine the specific localization of MP19 in the cell membrane and to determine whether the mutant MP19To3 protein migrates to the cell membrane in a similar fashion to normal MP19. METHODS: MP19 and MP19To3 cDNAs were cloned into two different sets of expression vectors. The first set was composed of two vectors, pEGFP-N1 and pDsRed2-N1. The first vector expressed green fluorescent protein and the second expressed a red fluorescent protein when transfected into mammalian cells. The two lens membrane protein cDNAs were separately cloned into the vectors so that the cDNA was at the 5'-end of the fluorescent protein coding DNA. These vectors expressed each of the lens proteins fused to the fluorescent protein upon transfection into mammalian cell cultures. The second vector set was a single vector, pcDNA4/TO which must be induced in the transfected cells by tetracycline in order to express the cloned cDNAs. Each of the membrane cDNAs coupled to the fluorescent protein coding region was cut out of the first vector set and cloned into pcDNA4/TO and stable clones were isolated. Each of the prepared plasmids was transfected into human and chick embryo lens epithelial cells and human T-RexTM-293 cells. The fluorescent cells were viewed using confocal and episcopic-fluorescence microscopy. RESULTS: Each of the transfected plasmids expressed fluorescent protein in all three cell lines. MP19 was observed to transport to the cell membrane. When compared to the distribution of another, separate fusion protein consisting of a signal peptide that targets to cell membranes fused to EGFP, MP19 did not distribute uniformly on the membrane, but appeared to localize into "spots" or pools of fluorescent material around the cell membrane. In contrast, MP19To3 protein appeared to not distribute to the cell membrane; it instead appeared to collect in a particular subcellular compartment within the cell. CONCLUSIONS: The distribution of MP19 and MP19To3 in the cell appeared to be quite distinct. MP19 was observed to distribute to the cell membrane while MP19To3 did not. The fact that the MP19To3 did not traffic to the membrane, instead appearing to be trapped within a subcellular compartment within the cell sheds further light on the cause of the cataract and microphthalmia observed in the MP19To3 mutation, and further sheds information on the pathway of MP19 transport to the cell membrane.

Animals↗
PubMed1995

Overexpression, purification, and characterization of Escherichia coli acyl carrier protein and two mutant proteins.

A synthetic gene of 237 bases encoding the 77-residue acyl carrier protein (ACP) from Escherichia coli, along with two mutant genes, ACP-I54V and ACP-A59V, were subcloned into the pET11a-pLysS E. coli overexpression system under the control of the bacteriophage T7 promoter. This efficient expression system and a simplified purification protocol yielded more than 120 mg/l of pure protein. The construct produced a mixture of holo-ACP and apo-ACP and two HPLC procedures were developed to separate the two species. This overexpression system allows cost-effective growths of 13C- and 15N-labeled protein for structural and other studies on ACP. In the course of the work on the mutants of ACP, an apparent homologous recombination event led, in one case, to reversion to a wild-type protein, suggesting that precautions to prevent such reversion should be taken.

Acyl Carrier Protein↗
PubMed1999

G33D mutant thioredoxin primarily affects the kinetics of reaction with thioredoxin reductase. Probing the structure of the mutant protein.

Escherichia coli thioredoxin is a redox-active protein. A mutant protein with an aspartic acid substitution for the largely conserved glycine at position 33 (G33D) in the active site of thioredoxin has been generated to study the effects of a negatively charged residue in the active site of the protein. Despite the close proximity of the negative-charged Asp to the redox active cysteines, the effective concentration of the cysteines does not deviate significantly from that of the wild-type protein. The redox potential (E(o)') measured by the equilibrium between NADPH and the mutant thioredoxin is also close to that of the wild-type. Kinetic measurements of the reaction between thioredoxin and thioredoxin reductase show that G33D mutant and the wild-type proteins have identical kcat values. However, the Km for G33D mutant is approximately 10-fold higher than that for the wild-type protein. In vivo assay of the growth of E. coli strain carrying wild-type or G33D mutant thioredoxin on methionine sulfoxide indicates that the G33D mutant protein is a slower electron donor for methionine sulfoxide reductase. Structural stability of the oxidized protein is not altered by the G33D substitution, as illustrated by the same unfolding free energies studied by urea. The substitution does not show significant change of the near UV and far-UV circular dichroic (CD) and the fluorescence spectra for either the reduced or the oxidized protein. Therefore, the global structure of the G33D protein is not changed. However, the surface of the active site has been altered locally by G33D substitution, which accounts for the above kinetically poor behaviors. A model of G33D structure is constructed based on these studies.

Aspartic Acid↗
PubMed1993

Concomitant loss of conformation and superantigenic activity of staphylococcal enterotoxin B deletion mutant proteins.

The T-cell-stimulating activity of staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases. To investigate the immunologically active domains of the SEB molecule, we have produced truncated fragments of recombinant SEB by C-terminal and N-terminal deletions. The fragments were expressed as fusion proteins with protein A, including a cleavage site to remove the protein A part. Mutant proteins were tested for the ability to stimulate human resting T cells and SEB-reactive T-cell clones. Deletion of only 9 amino acids from the C terminus leads to complete loss of T-cell-stimulating activity. Removing further amino acids from the SEB molecule did not lead to a reexpression of T-cell-mitogenic activity. A mutant protein, however, in which the 9 C-terminal amino acids were replaced with a tail of 68 amino acids encoded by the vector was fully active. Two mutant proteins with N-terminal deletions of 60 and 81 amino acids were inactive as well. A neutralizing monoclonal antibody against a conformational epitope lost binding with all the inactive mutant proteins only, whereas a monoclonal antibody recognizing an epitope involved in emetic activity reacted with all mutant proteins. These results suggest that even small deletions at the C terminus affect the three-dimensional conformation of the SEB molecule.

Amino Acid Sequence↗
PubMed1999

Computational studies on mutant protein stability: The correlation between surface thermal expansion and protein stability.

Thermal stability of mutant proteins has been investigated using temperature dependent molecular dynamics (MD) simulations in vacuo. The numerical modeling was aimed at mimicking protein expansion upon heating. After the conditions for an expanding protein accessible surface area were established for T4 lysozyme and barnase wild-type proteins, MD simulations were carried out under the same conditions using the crystal structures of several mutant proteins. The computed thermal expansion of the accessible surface area of mutant proteins was found to be strongly correlated with their experimentally measured stabilities. A similar, albeit weaker, correlation was observed for model mutant proteins. This opens the possibility of obtaining stability information directly from protein structure.

Bacterial Proteins↗
PubMed1991

Novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein: enzymatic analysis of the mutant proteins.

Using the techniques of gap misrepair mutagenesis and site-directed mutagenesis, we have generated two novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein. DNA sequencing showed these mutations to be Ser-83----Ala and Gln-106----Arg. The mutant proteins were overproduced and purified, and their enzymatic properties were analyzed and compared with those of the wild-type enzyme. With ciprofloxacin and other quinolones, the inhibition of DNA supercoiling, relaxation, and decatenation and the induction of DNA cleavage were investigated for both wild-type and mutant enzymes. In each assay, the mutant enzymes were found to require approximately 10 times more drug to inhibit the reaction or induce cleavage than was the wild-type enzyme. However, the Ca2(+)-directed DNA cleavage reaction was indistinguishable for wild-type and mutant gyrases. We discuss models for the gyrase-mediated bactericidal effects of quinolone drugs.

4-Quinolones↗
PubMed1984

The antibody response to a single antigenic determinant of the tobacco mosaic virus protein: analysis using monoclonal antibodies, mutant proteins and synthetic peptides.

Three hybridomas were selected which secreted monoclonal antibodies specific to a decapeptide determinant representing residues 103-112 of the tobacco mosaic virus protein ( TMVP ). A series of proteins from several strains of TMV which differ in the amino acid sequence in this region of the protein were used as probes for specificity analysis. The fine-specificity analysis was extended by assessing the binding of the antibodies with a panel of synthetic peptide analogues of the native decapeptide with amino acid substitutions at different locations. The binding of each synthetic peptide with each of the monoclonal antibodies was determined by the ability of the radiolabeled peptide to bind with the antibody. The binding of the decapeptide with antibodies was determined by equilibrium dialysis; the relative binding affinity of each peptide of the panel was determined by the capacity of the peptide to inhibit the binding between the antibody and the radiolabeled native decapeptide. The results demonstrated that a panel of synthetic peptide analogues constitutes a powerful tool for discerning the fine specificity of antibodies directed to a given determinant of a protein antigen. The data indicated that, although all of the antibodies recognized the same nominal decapeptide determinant, their binding with the different mutant proteins or with the synthetic peptides of the panel differed greatly, indicating dramatic differences in their fine specificity. The existence of such differences should be taken into consideration when assessing residues of a protein antigen that are involved in antibody binding. The differences which were found in monoclonal antibodies produced following immunization with the whole TMVP reflect differences which occur in heterogeneous serum antibody populations and point out the complexity of antigenic recognition even of as small an epitope as a decapeptide.

Amino Acid Sequence↗
PubMed1977

Improved electrophoretic and immunochemical techniques for the identification and characterization of mutant proteins, applied to ribosomal protein S8 in Escherichia coli mutants.

The ribosomal proteins of 11 mutants which are sensitive to starvation at elevated temperature and of 36 transductants derived from them were studied with several electrophoretic, immunochemical and proteinchemical methods. The following results were obtained: (1) Ribosomal protein S8 is altered in three of these mutants. (2) The amino acid exchange in proteins S8 of mutant N4128 is Glu leads to Lys in position 59 of the protein chain. (3) Temperature sensitivity and inability to recover from starvation at elevated temperatures are caused by the same mutational event which is, however, unrelated to the alteration in protein S8. Several electrophoretic and immunological procedures were applied during the characterization of these mutants. A modified immunoelectrophoresis on cellulose acetate gels was developed, and proved to be the most applicable procedure for the detection of mutationally altered ribosomal proteins. This procedure may gain general importance for detecting mutational alterations in other proteins.

Amino Acid Sequence↗
PubMed1994

A versatile expression vector for the in vitro study of protein-protein interactions: characterization of E47 mutant proteins.

Several mutants of the E47 protein, a member of the family of basic/helix-loop-helix (b-HLH) transcriptional regulators, were examined for their ability to homo- and heterodimerize with the protein product of the T-cell oncogene tal-1/SCL. For this purpose, a novel bacterial expression system was developed in which proteins are expressed as fusions appended to glutathione-S-transferase via a thrombin cleavage site and either one or four protein kinase recognition sites embedded in a glycine-rich domain. Since the interaction domain can be purified away from the glutathione-S-transferase moiety and the radioactive label is located in a flexible N-terminal tag, protein folding should occur normally. Our studies with E47 proteins prepared in this system indicate that the ratio between E47 homodimers and E47/tal-1 heterodimers can dramatically shift upon subtle mutations in the loop region and the second helix of the E47 protein. This unexpected results suggests a novel mechanism to alter the equilibrium between different transactivating protein complexes of the b-HLH class.

Amino Acid Sequence↗
PubMed1990

Disruption of the actin cytoskeleton in yeast capping protein mutants.

Capping protein controls the addition of actin subunits to the barbed end of actin filaments and nucleates actin polymerization in vitro. Capping protein has been identified in all eukaryotic cells examined so far; it is a heterodimer with subunits of relative molecular masses 32,000-36,000 (alpha-subunit) and 28,000-32,000 (beta-subunit). In skeletal muscle, capping protein (CapZ) probably binds the barbed ends of actin filaments at the Z line. The in vivo role of this protein in non-muscle cells is not known. We report here the characterization of CAP2, the single gene encoding the beta-subunit of capping protein in Saccharomyces cerevisiae. Yeast cells in which the CAP2 gene was disrupted by an insertion or a deletion had an abnormal actin distribution, including the loss of actin cables. The mutant cells were round and large, with a heterogeneous size distribution, and, although viable, grew more slowly than congenic wild-type cells. Chitin, a cell wall component restricted to the mother-bud junction in wild-type budding yeast, was found on the entire mother cell surface in the mutants. The phenotype of CAP2 disruption resembled that of temperature-sensitive mutations in the yeast actin gene ACT1, indicating that capping protein regulates actin-filament distribution in vivo.

Actin Depolymerizing Factors↗
PubMed1999

The Protein Mutant Database.

Currently the protein mutant database (PMD) contains over 81 000 mutants, including artificial as well as natural mutants of various proteins extracted from about 10 000 articles. We recently developed a powerful viewing and retrieving system (http://pmd.ddbj.nig.ac.jp), which is integrated with the sequence and tertiary structure databases. The system has the following features: (i) mutated sequences are displayed after being automatically generated from the information described in the entry together with the sequence data of wild-type proteins integrated. This is a convenient feature because it allows one to see the position of altered amino acids (shown in a different color) in the entire sequence of a wild-type protein; (ii) for those proteins whose 3D structures have been experimentally determined, a 3D structure is displayed to show mutation sites in a different color; (iii) a sequence homology search against PMD can be carried out with any query sequence; (iv) a summary of mutations of homologous sequences can be displayed, which shows all the mutations at a certain site of a protein, recorded throughout the PMD.

Amino Acid Sequence↗