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PubMed1996

Analysis of allelic structures at the D7S21 (MS31A) locus in the Japanese, using minisatellite variant repeat mapping by PCR (MVR-PCR).

To sample the diversity of allelic structures at the D7821 (MS31 A) locus in the Japanese, allele-specific minisatellite variant repeat mapping using polymerase chain reaction (MVR-PCR) was performed on genomic DNA from a number of Japanese individuals. Three polymorphic positions in the MS31A 5' flanking DNA were typed from 214 un related Japanese, and the distribution of haplotypes was analysed. Allele-specific MVR-PCR using primers that discriminate between different alleles at these polymorphic positions in heterozygous individuals, allows single alleles to be mapped from genomic DNA in approximately 80% of Japanese. 149 Japanese alleles have been mapped to date and all of them, except for two pairs of indistinguishable alleles, have different internal structures. More than half of the mapped alleles showed similar regions of internal structure to other alleles and were classified into groups on this basis.

Alleles↗
PubMed1991

Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c.

The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR. The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein. In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved. Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38. The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3188-3197] appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification.

Alanine↗
PubMed1988

Plant small nuclear RNAs. V. U4 RNA is present in broad bean plants in the form of sequence variants and is base-paired with U6 RNA.

U4 RNA, which is known to play an indispensable role in pre-mRNA splicing, is present in plant nuclei, has a canonical m3 2,2,7 G cap at its 5' end and is associated with U6 RNA in snRNP particles. It occurs in broad bean in the form of a number of sequence variants. Two of these were sequenced: U4A RNA is 154 and U4B RNA is 152 nucleotides long. Sequence similarity of broad bean U4B RNA is 94 per cent to broad bean U4A RNA, 65 per cent to rat U4A RNA, 61 per cent to Drosophila U4A RNA and 50 per cent to snR14, the U4 RNA equivalent of the yeast Saccharomyces cerevisiae. Sequence conservation is much more pronounced in the 5' half of the molecule than in its 3' half. The secondary structure of both variants of broad bean U4 RNA perfectly fits with that of all other U4 RNAs sequenced so far. Nucleotide changes between broad bean U4A and U4B RNAs are restricted to molecular regions that affect the thermodynamic stability of these molecules. A model is proposed for the base pairing interaction of broad bean U4 RNA with broad bean U6 RNA. This is the first report on the structure of a plant U4 RNA.

Animals↗
PubMed1995

Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation.

Amino Acid Sequence↗
PubMed2003

Evaluation of cooperative interactions between substructures of iso-1-cytochrome c using double mutant cycles.

A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 --> isoleucine (N52I) in iso-1-cytochrome c and mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100. These histidine mutation sites are distributed through the four cooperative folding units of cytochrome c. The double mutant cycle starts with the iso-1-cytochrome c variant AcTM, a variant with no surface histidines and with asparagine at position 52. Isoleucine is added singly at position 52, AcTMI52 variant, as are the surface histidines, AcHX variants, where X indicates the histidine sequence position. The double mutant variants, AcHXI52, provide the remaining corner of the double mutant cycle. The stabilities of all variants were determined by guanidine hydrochloride denaturation and interaction energies were calculated between position 52 and each histidine site. Six of the seven double mutants show additive (AcH33I52, AcH39I52, AcH54I52, AcH89I52, and AcH100I52) stability effects or weak interaction energies (AcH73I52) of the histidine mutations and the N52I mutation, consistent with cooperative effects on protein folding and stability being sparsely distributed through the protein structure. The AcH26I52 variant shows a strong favorable interaction energy, 2.0 +/- 0.5 kcal/mol, between the N52I mutation in one substructure and the addition of His 26 to an adjacent substructure. The data are consistent with an entropic stabilization of the intersubstructure hydrogen bond between His 26 and Glu 44 by the Ile 52 mutation.

Amino Acid Substitution↗
PubMed1992

Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver, epithelial and endothelial cells. Structurally, it has similarities to kringle-containing serine proteases, although it does not possess proteolytic activity. A structure-activity relationship study of human HGF was performed by functional analysis of HGF substitution and deletion variants. Analysis of HGF variants was accomplished by defining their ability to induce DNA synthesis on hepatocytes in primary culture and to compete with wild-type HGF for binding to a soluble form of the HGF receptor. Three groups of variants were made: (i) substitutions at the cleavage site, (ii) substitutions within the protease-like domain and (iii) deletions of the beta-chain and/or kringle domains. Our results show that: (i) single-chain HGF is a zymogen-like promitogen in that cleavage into a two-chain form is required for biological activity, however, the single chain form of HGF still retains substantial receptor binding capacity; (ii) certain mutations in the protease-like domain result in variants that are completely defective for mitogenic activity, yet exhibit apparent receptor binding affinities similar to wild-type HGF (Kd approximately 50-70 pM); and (iii) a variant containing the N-terminal 272 residues of mature HGF showed only a 4-fold increase in Kd when compared with wild-type HGF indicating that a primary receptor binding determinant is located within this sequence.

Animals↗
PubMed1984

Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes.

It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.

Amino Acid Sequence↗
PubMed1993

Organization and evolution of an alpha satellite DNA subset shared by human chromosomes 13 and 21.

The structure of the alpha satellite DNA higher-order repeat (HOR) unit from a subset shared by human chromosomes 13 and 21 (D13Z1 and D21Z1) has been examined in detail. By using a panel of hybrids possessing either a chromosome 13 or a chromosome 21, different HOR unit genotypes on chromosomes 13 and 21 have been distinguished. We have also determined the basis for a variant HOR unit structure found on approximately 8% of chromosomes 13 but not at all on chromosomes 21. Genomic restriction maps of the HOR units found on the two chromosome 13 genotypes and on the chromosome 21 genotype are constructed and compared. The nucleotide sequence of a predominant 1.9-kilobasepair HOR unit from the D13Z1/D21Z1 subset has been determined. The DNA sequences of different alpha satellite monomers comprising the HOR are compared, and the data are used to develop a model, based on unequal crossing-over, for the evolution of the current HOR unit found at the centromeres of both these chromosomes.

Animals↗
PubMed2000

The streptococcal superantigen SMEZ exhibits wide allelic variation, mosaic structure, and significant antigenic variation.

The frequencies of the newly identified streptococcal superantigen genes smez, spe-g, and spe-h were determined in a panel of 103 clinical isolates collected between 1976 and 1998 at various locations throughout New Zealand. smez and spe-g were found in every group A Streptococcus (GAS) isolate, suggesting a chromosomal location. The spe-h gene was found in only 24% of the GAS isolates and is probably located on a mobile DNA element. The smez gene displays extensive allelic variation and appears to be in linkage equilibrium with the M/emm type. 22 novel smez alleles were identified from 21 different M/emm types in addition to the already reported alleles smez and smez-2 with sequence identities between 94. 5 and 99.9%. Three alleles are nonfunctional due to a single base pair deletion. The remaining 21 alleles encode distinct SMEZ variants. The mosaic structure of the smez gene suggests that this polymorphism has arisen from homologous recombination events rather than random point mutation. The recently resolved SMEZ-2 crystal structure shows that the polymorphic residues are mainly surface exposed and scattered over the entire protein. The allelic variation did not affect either Vbeta specificity or potency, but did result in significant antigenic differences. Neutralizing antibody responses of individual human sera against different SMEZ variants varied significantly. 98% of sera completely neutralized SMEZ-1, but only 85% neutralized SMEZ-2, a very potent variant that has not yet been found in any New Zealand isolate. SMEZ-specific Vbeta8 activity was found in culture supernatants of 66% of the GAS isolates, indicating a potential base for the development of a SMEZ targeting vaccine.

Alleles↗
PubMed1985

[Immunomorphological study of the distribution of prekeratin with a molecular weight of 49 kilodaltons in various epithelial cells in rats].

Cryostat sections of various tissues of rat were stained using an indirect immunofluorescent method with monoclonal antibody against individual prekeratin with the molecular mass of 49 kilodalton (PK-49). Connective tissue endothelial cells, neurons, glia, haematopoetic tissue and smooth muscles were completely negative in this test. 46 morphological variants of epithelial structures were investigated. PK-49 was absent from all the stratified epithelia (epidermis, hair folliculi, oesophagus) but was expressed in virtually all simple epithelia of endodermal origin (exceptions: squamous lung alveolar epithelium and germinative epithelium of testis). There were negative (kidney tubules) as well as positive (bladder, mammary, glands) cell elements among mesodermal and ectodermal simple epithelia. High specificity of individual PK in respect to morphological variants of epithelia points out to the important role played by prekeratin-type intermediate filaments in morphogenesis.

Animals↗
PubMed2002

2.8 A crystal structures of recombinant fibrinogen fragment D with and without two peptide ligands: GHRP binding to the "b" site disrupts its nearby calcium-binding site.

We report two crystal structures, each at a resolution of 2.8 A, of recombinant human fibrinogen fragment D (rfD) in the absence and presence of peptide ligands. The bound ligands, Gly-Pro-Arg-Pro-amide and Gly-His-Arg-Pro-amide, mimic the interactions of the thrombin exposed polymerization sites, "A" and "B", respectively. This report is the first to describe the structure of fragment D in the presence of both peptide ligands. The structures reveal that recombinant fibrinogen is nearly identical to the plasma protein but with minor changes, like the addition of a proximal fucose to the carbohydrate linked to residue betaGln364, and slightly different relative positions of the beta- and gamma-modules. Of major interest in our structures is that a previously identified calcium site in plasma fibrinogen is absent when Gly-His-Arg-Pro-amide is bound. The peptide-dependent loss of this calcium site may have significant biological implications that are further discussed. These structures provide a foundation for the detailed structural analysis of variant recombinant fibrinogens that were used to identify critical functional residues within fragment D.

Animals↗
PubMed1974

Genetic limits of the xanthine dehydrogenase structural element within the rosy locus in Drosophila melanogaster.

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.

Aldehyde Oxidoreductases↗
PubMed1997

Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants.

A battery of relatively simple tests allows the presumptive identification of hemoglobin (Hb) variants, making unnecessary structural analysis by protein chemistry methods or DNA sequencing. The primary step in this strategy involves the use of a matrix of electrophoretic mobilities obtained under various experimental conditions. This leads to an unambiguous result in approximately 90% of the cases. Additional tests are required to characterize with more confidence the remaining 10%. We describe here the use of cation-exchange HPLC on the Bio-Rad Variant automated analyzer with the "beta Thalassemia Short" program. By comparing the elution time of 125 human Hb mutants, we found that some variants with almost identical pI values or produced by the same type of amino acid substitution displayed different elution times. We present several examples in which use of the HPLC profile helped establish the diagnosis.

Cations↗
PubMed2006

Determination of unique amino acid substitutions in protein variants by peptide mass mapping with FT-ICR MS.

Peptide mass mapping plays a central role in the structural characterization of protein variants with single amino acid substitutions. Among the 20 standard amino acids found in living organisms, 18, all but Leu and Ile, differ from each other in molecular mass. The mass differences between amino acids range from 0.0364 to 129.0578 Da. The mass of the mutated peptide or the difference between normal and mutated peptides uniquely determines the type of substitution in some cases, and even pinpoints the position of the mutation when the involved residue is found only once in the peptide. Among 75 pairs of amino acid residues that are exchangeable via a single nucleotide replacement, 53 show specific change in exact mass, while only 25 in nominal mass. On the other hand, precise measurement, at least to the third decimal place, greatly enhances the capacity of the peptide mass mapping strategy for structural characterization. This notion was verified by an analysis of three Hb variants using MALDI-FTICR MS. In addition, the baseline resolution of two 1 kDa peptides with a single amino acid difference, Lys or Gln, which have the smallest (0.0364 Da) difference among residues, was achieved by measurement at a mass resolving power of 342,000. The results indicated that the smallest difference, 0.0040 Da between [Delta29.9742 for Glu-Val] and [Delta29.9782 for Trp-Arg], among all types of amino acid substitutions derived from a single nucleotide replacement can be discriminated at the present performance level. Therefore, FTICR MS is capable of identifying all 53 types of substitutions, each of which is associated with a unique mass difference, except for the Leu and Ile isomers.

Amino Acid Sequence↗
PubMed1993

Further structural definition of a new family of glycopeptidolipids from Mycobacterium xenopi.

The highly antigenic surface glycolipids of serovars of the Mycobacterium avium complex are glycopeptidolipids of the general structure [formula: see text] However, it has recently been shown [Rivière, M., & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063; Rivière, M., & Puzo, G. (1992) Biochemistry 31, 3575-3580] that the characteristic glycopeptidolipid of a strain of Mycobacterium xenopi is of the structure [formula: see text] [monosaccharide, 3-O-CH3-6-deoxy-alpha-L-talopyranose; tetrasaccharide, 2,3,4-tri-O-CH3-alpha-L-rhamnopyranose(1-->3)2-O-lauryl-alpha-L- rhamnopyranose(1-->3)2,4-di-O-acetyl/lauryl-6-deoxy-alpha-L-glucopyranos e]. The type of analyses previously applied to the glycopeptidolipids of M. avium combined with fast atom bombardment mass spectrometry allowed the recognition of other variants of this structure in other strains of M. xenopi: [formula: see text] in which the monosaccharide is 2-O-acetyl-3-O-CH3-6-deoxy-alpha-L-talopyranose or 3-O-CH3-6-deoxy-alpha-L-talopyranose, and the disaccharide is 4-O-octanoyl(or decanoyl)-alpha-L- rhamnopyranose(1-->3)2-O-lauryl-alpha-L-rhamnopyranose or alpha-L-rhamnopyranose(1-->3)2-O-lauryl-alpha-L-rhamnopyranose. Recognition of serine-containing glycopeptidolipids substituted in one case with a tetrasaccharide and in another case with a disaccharide unit implies that M. xenopi, an important opportunistic pathogen, exists as a serocomplex in nature.

Amino Acid Sequence↗
PubMed2006

Design of a hyperstable protein by rational consideration of unfolded state interactions.

Stabilization of proteins is a long-sought objective. Targeting the unfolded state interactions of a protein is not a method used for this purpose, although many proteins are known to contain such interactions. The N-terminal domain of ribosomal protein L9 (NTL9) has a lysine residue at position 12, which makes strong non-native interactions in the unfolded state. Substitution of a d-alanine for G34 in NTL9 is known to stabilize the protein by reducing the entropy of the unfolded state. Here we combine these two mutations to design a hyperstable protein. The structure of the variant is the same as that of wild-type as judged by 2D NMR. The variant is hyperstable as judged by denaturation experiments, where complete thermal unfolding of the protein does not occur in native buffer.

Alanine↗
PubMed2004

Population structure and genetic variability within isolates of Grapevine fanleaf virus from a naturally infected vineyard in France: evidence for mixed infection and recombination.

The nematode-borne Grapevine fanleaf virus, from the genus Nepovirus in the family Comoviridae, causes severe degeneration of grapevines in most vineyards worldwide. We characterized 347 isolates from transgenic and conventional grapevines from two vineyard sites in the Champagne region of France for their molecular variant composition. The population structure and genetic diversity were examined in the coat protein gene by IC-RT-PCR-RFLP analysis with EcoRI and StyI, and nucleotide sequencing, respectively. RFLP data suggested that 55 % (191 of 347) of the isolates had a population structure consisting of one predominant variant. Sequencing data of 51 isolates representing the different restrictotypes confirmed the existence of mixed infection with a frequency of 33 % (17 of 51) and showed two major predominant haplotypes representing 71 % (60 of 85) of the sequence variants. Comparative nucleotide diversity among population subsets implied a lack of genetic differentiation according to host (transgenic vs conventional) or field site for most restrictotypes (17 of 18 and 13 of 18) and for haplotypes in most phylogenetic groups (seven of eight and six of eight), respectively. Interestingly, five of the 85 haplotypes sequenced had an intermediate divergence (0.036-0.066) between the lower (0.005-0.028) and upper range (0.083-0.138) of nucleotide variability, suggesting the occurrence of homologous RNA recombination. Sequence alignments clearly indicated a mosaic structure for four of these five variants, for which recombination sites were identified and parental lineages proposed. This is the first in-depth characterization of the population structure and genetic diversity in a nepovirus.

Base Sequence↗