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PubMed1993

The structures of native phosphorylated chicken cystatin and of a recombinant unphosphorylated variant in solution.

The solution structures of the phosphorylated form of native chicken cystatin and the recombinant variant AEF-S1M-M29I-M89L were determined by 2D, 3D and 4D-NMR. The structures turn out to be very similar, despite the substitutions and the phosphorylation of the wild-type. Their dominant feature is a five-stranded beta-sheet, which is wrapped around a five-turn alpha-helix, as shown by X-ray crystallographic studies of wild-type chicken cystatin. However, the NMR analysis shows that the second helix observed in the crystal is not present in solution. The phosphorylation occurs at S80, which is located in a flexible region. For this reason, very few effects on the structure are observed. Comparison of structures of the unphosphorylated variant and the wild-type shows small effects on H84 which is located in the supposed recognition site of the serine kinase. This recognition site appears to be well structured as a large loop-containing bulge of the beta-sheet. The N termini of both mutants, which contribute to a large extent to the binding to the proteinase, are very flexible. A loop structure involving the residues L7 to A10 as found in related inhibitors, such as in the kininogen domains 2 and 3, is not sufficiently populated to be observed.

Amino Acid Sequence↗
PubMed1990

Distinctive structure and function of human apolipoprotein variant ApoA-IV-2.

We have investigated the molecular structure, phospholipid binding, and lecithin-cholesterol acyltransferase catalytic activity of pure apoA-IV-2, a basic variant isoform of apoA-IV which is inherited as a classical Mendelian allele with a gene frequency of 0.09. Circular dichroism spectroscopy established that the alpha-helical content of apoA-IV-2 was 75% in the native state (versus 56% for apoA-IV-1), and increased to 88% in the presence of phospholipid. Fluorescence titration established that apoA-IV-2 bound to egg phospholipid vesicles with a Ka of 3.3 x 10(6) liter/mol, 2.4-fold greater than the affinity of apoA-IV-1. Fluorescence quenching studies revealed that, unlike apoA-IV-1, binding of apoA-IV-2 to phospholipid vesicles induced strong shielding of the amino-terminal tryptophan against iodide quenching. Enzyme kinetic studies using both saturated and unsaturated phospholipid substrates demonstrated that apoA-IV-2 was 36-71% more efficient in activating lecithin-cholesterol acyltransferase than apoA-IV-1. We conclude that apoA-IV-2 has more alpha-helical structure, is more stable in solution, and is more hydrophobic than apoA-IV-1, and that these distinctive structural features are associated with a higher affinity for phospholipid surfaces and an increased catalytic efficiency of lecithin:cholesterol acyltransferase activation. The biophysical basis for this latter characteristic may be the ability of apoA-IV-2 to penetrate phospholipid surfaces to a greater depth than apoA-IV-1. These molecular properties may be responsible for the increased levels of high density lipoproteins which have been observed in apoA-IV-2 heterozygotes.

Alleles↗
PubMed1982

Human histocompatibility antigen mutants immunoselected in vitro. Biochemical analysis of a mutant which synthesizes an altered HLA-A2 heavy chain.

Immunoselection with HLA-A2 or HLA-A1 specific alloantisera has been utilized to isolate spontaneously arising and mutagen-induced variants from the B lymphoblastoid cell line T5-1 (HLA haplotypes DR3, B8, A1 and DR1, B27, Cw1, A2). Such variants are characterized by reduced reactivity with alloantisera of the selecting specificity, but normal reactivity with alloantisera directed to the other HLA specificities of T5-1. Biochemical analysis reveals two classes of variants. In all HLA-A1 and some HLA-A2 variants, the heavy chain in question cannot be detected; however, in other HLA-A2 variants, a structurally altered HLA-A2 heavy chain is found. In the HLA-A2 variant 6.6.5, this heavy chain is glycosylated and thus has presumably been inserted into the rough endoplasmic reticulum membrane in vivo. However, unlike all other HLA heavy chains, the 6.6.5 HLA-A2 heavy chain does not associate with beta 2-microglobulin, does not undergo processing of its high mannose oligosaccharide, and does not migrate to the cell surface, although it is relatively stably expressed within the cell. We suggest that the primary defect in these cells is the failure of the 6.6.5 HLA-A2 heavy chain to associate stably with beta 2-microglobulin. It is likely that the observed structural alteration in this heavy chain reflects a change in amino acid sequence, and thus, a mutation in the structural gene encoding HLA-A2.

Cell Line↗
PubMed1981

Structural analysis of human hemoglobin variants with field desorption mass spectrometry.

Tryptic hydrolyzates of normal and abnormal human globin chains were analyzed with mass spectrometry, using field desorption ionization technique. All the peptides, including core peptides, were detected as protonated molecular ions in field desorption mass spectra. Since the technique makes it possible to determine the mass number of each peptide, it is very useful for structural analysis of human hemoglobin variants, even those with electrophoretically and chromatographically silent mutations.

Adult↗
PubMed2026

A De Novo 16p13.3 Triplication Underlying Early-Onset Complex Neurodegeneration.

BACKGROUND: Neurodegenerative disorders are clinically and genetically heterogeneous, characterized by progressive neuronal loss and multidomain functional decline. Despite a presumed genetic etiology, a substantial proportion of cases remain molecularly undiagnosed. OBJECTIVE: The aim was to identify the genetic cause of an early-onset neurodegenerative disorder presenting with ataxia and cognitive impairment. METHODS: Rare copy-number variants were detected via short-read whole-genome sequencing (WGS), with candidate structural models inferred using long-read WGS. We performed transcriptomic profiling of peripheral blood leukocytes by RNA sequencing, with validation using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: We identified a de novo copy-number gain at 16p13.3. Combined copy-number profiling and long-read WGS suggested a candidate model comprising a triplicated segment in tandem with a proximal duplication, joined to a distal duplication via an inverted junction. Transcriptomic analysis demonstrated significant upregulation of ATP6V0C, AMDHD2, and PDPK1. CONCLUSIONS: These findings support a role for structural variation in early-onset neurodegeneration and highlight the value of combining short-read copy-number profiling with long-read WGS to detect and characterize complex genomic rearrangements. © 2026 International Parkinson and Movement Disorder Society.

16p13.3↗
PubMedDate not supplied

[Tissue specificity of alternative splicing products of mouse mRNA encoding new protein hampin homologous to the Drosophila MSL-1 protein].

A number of mammalian genomes have one gene copy encoding the protein that we named hampin. A search in a number of databases revealed a distant homologue, the well-known Drosophila protein MSL-1 (male-specific lethal 1). An alternative splicing of mRNA led to a significant diversity of structural hampin variants with different domain compositions. We analyzed the tissue-specific expression of five mouse hampin variants using RT-PCR. Two variants encoding hampin proteins with truncated N termini were shown to have a restricted tissue specificity: they are exclusively expressed in the testes. The mRNAs of other hampin variants were detected in all the tested tissues at comparable levels. We obtained polyclonal antibodies to the recombinant hampin and used them to demonstrate that at least one of the variants is predominantly localized in the nucleus. The specific features of the hampin primary structure and its possible functions as a member of the hampin/MSL-1 family of proteins are discussed.

Alternative Splicing↗
PubMed1990

Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutations at the beta 27 (B9) position.

We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the beta chains, namely, Hb Knossos or beta A27S and Hb Grange-Blanche or beta A27V. The beta A27S mutant has a 50% decrease in oxygen affinity relative to native human Hb A, while the beta A27V mutant has an increased oxygen affinity. We have also engineered the artificial beta A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of Hb A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the beta chains; for the beta A27S mutant a new hydrogen bond between serine and the carbonyl O at beta 23 (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native Hb A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities.

Binding Sites↗
PubMedDate not supplied

Molecular analysis of different allelic variants of wild-type human p53.

The p53 tumour suppressor gene is intensively studied because mutations in this gene are the most common genetic alteration so far identified in human cancer. Considerable emphasis has thus been placed on characterizing the biological differences between mutant and wild-type p53 protein. This has led to the realization that in cultured cells, mutant p53 behaves like an oncogene, whereas wild-type p53 is a tumor suppressor gene. The p53 protein is also a target for the tumour virus oncogene products SV40 large T, adenovirus E1B, and human papillomavirus type 16 E6, which are all capable of forming complexes to the p53 protein. Although p53 represents an extremely important cellular regulatory molecule which is well conserved, there exists two allelic variants of wild-type human p53 that differ both in primary and confirmational structure. One variant contains an arginine at amino acid 72 (p53Arg), whereas the other form contains a proline at this residue (p53Pro). The possible implications for more than one allelic variant of wild-type human p53 in the general population is unknown. The present study was undertaken to compare some of the biological features of the different wild-type p53 variants. We present data demonstrating that there was a post-transcriptional selection against accumulation of both variants of wild-type human p53 in 3T3-A31 cells, arguing that both forms are proliferation inhibitory in these cells. Both variants of human p53 were stabilized by SV40 large T, but did not displace mouse p53 from SV40 large T. Neither allelic variant of human p53 was able to reduce significantly SV40-mediated anchorage-independent growth of 3T3-A31 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1993

Separation and tryptic digest mapping of normal and variant haemoglobins by capillary electrophoresis.

Characterization of haemoglobin (Hb) through whole protein separations and tryptic digest mapping allows the identification of structural Hb variants which result in haemoglobinopathies. Tryptic digest mapping by conventional two-dimensional paper chromatograph-electrophoresis provides high resolution but requires 48 h, while gradient elution reversed-phase high-performance liquid chromatography (HPLC) is faster (1.5 h), there is decreased resolution. CE analysis provides a fast separation with high resolution. We have used CE to optimise the tryptic digestion of globin purified from normal human haemoglobin A and to analyze tryptic digests from normal Hb. The separations were optimised and peak identification performed using UV scanning detection. In the optimised tryptic digest separation up to 28 peaks could be resolved in < 20 min. These peaks were identified as far as possible and a high-resolution map of the digest was constructed. The optimised analytical conditions were used to observe the separation pattern obtained from normal adult haemoglobin (HbA), common variant haemoglobins and some rarer haemoglobin variants.

Adult↗
PubMed2024

Identification of four TTN variants in three families with fetal akinesia deformation sequence.

BACKGROUND: TTN is a complex gene with large genomic size and highly repetitive structure. Pathogenic variants in TTN have been reported to cause a range of skeletal muscle and cardiac disorders. Homozygous or compound heterozygous mutations tend to cause a wide spectrum of phenotypes with congenital or childhood onset. The onset and severity of the features were considered to be correlated with the types and location of the TTN variants. METHODS: Whole-exome sequencing was performed on three unrelated families presenting with fetal akinesia deformation sequence (FADS), mainly characterized by reduced fetal movements and limb contractures. Sanger sequencing was performed to confirm the variants. RT-PCR analysis was performed. RESULTS: TTN c.38,876-2&#xa0;A&#x2009;>&#x2009;C, a meta transcript-only variant, with a second pathogenic or likely pathogenic variant in trans, was observed in five affected fetuses from the three families. Sanger sequencing showed that all the fetal variants were inherited from the parents. RT-PCR analysis showed two kinds of abnormal splicing, including intron 199 extension and skipping of 8 bases. CONCLUSIONS: Here we report on three unrelated families presenting with FADS caused by four TTN variants. In addition, our study demonstrates that pathogenic meta transcript-only TTN variant can lead to defects which is recognizable prenatally in a recessive manner.

Humans↗
PubMed1996

SpFGFR, a new member of the fibroblast growth factor receptor family, is developmentally regulated during early sea urchin development.

We describe the cloning of a new fibroblast growth factor receptor, SpFGFR1, that is differentially regulated at the level of transcript abundance during sea urchin embryogenesis. Sequence representing the conserved tyrosine kinase domain was obtained by reverse transcription-polymerase chain reaction using degenerate primers, and the entire open reading frame was obtained by standard cDNA library screening methods. SpFGFR contains a series of domains characteristic of FGFRs: three immunoglobulin-like motifs, an acid box, a transmembrane domain, a relatively long juxtamembrane sequence, a split tyrosine kinase domain, and two conserved intracellular tyrosine residues. Alternative splicing of SpFGFR generates two variants (Ig3L and Ig3S), which differ by insertion in the center of the Ig3 domain of 34 extra amino acids, encoded by an additional exon. Transcripts encoding both variants accumulate when morphogenesis begins with mesenchyme cell ingression and gastrulation. SpFGFR transcripts accumulate in all cell types of the embryo, although in situ hybridization shows that they are somewhat enriched in cells of oral ectoderm and endoderm. Transcripts encoding the Ig3S variant, whose structure resembles more closely that of vertebrate receptors, are enriched in endomesoderm, suggesting that the SpFGFR variants could play distinct roles in the sea urchin embryo.

Alternative Splicing↗
PubMed2002

Molecular characterization of Hb D-Ibadan [beta87(F3)Thr-->Lys] in combination with Hb S [beta6(A3)Glu-->Val] and with beta+-Thalassemia: report of two cases.

Hb D-Ibadan [beta87(F3)Thr-->Lys] is a common variant in the Nigerian population, which has been reported in association with Hb S [beta6(A3)Glu-->Val] and with beta-thalassemia. Unlike the Hb S/Hb D-Los Angeles [beta121(GH4)Glu-->Gln] combination, compound heterozygosity for Hb D-Ibadan and Hb S does not result in a sickling disorder. We report the first case of a combination of Hb D-Ibadan with beta+-thalassemia, and the first observation of Hb S/Hb D-Ibadan in the African-American population. In both cases, the characterization of Hb D-Ibadan was achieved by sequencing of the genomic DNA. Although protein based methods such as isoelec-trofocusing and high performance liquid chromatography may suggest that the "D-like" variant is different from Hb D-Los Angeles, the definitive identification of the variant by structural analysis or molecular genetic methods should be undertaken, particularly in newborn screening programs when the variant is found in combination with Hb S.

Adult↗
PubMed1990

Topological analysis of hydrogen bonding in protein structure.

A recent study has shown that topological stereoisomers exist for the polypeptide chain in disulfide-containing proteins that are represented by non-planar graphs. This topological stereochemistry is demonstrated in the structure of variant 3 toxin in the venom of the North American scorpion Centruroides sculpturatus Ewing and the structure of toxin II from the North African scorpion Androctonus australis Hector. In this report, we found that a similar topological analysis can be applied to the hydrogen bonding in alpha-helices and beta-sheets within protein molecules, and we described the topological characteristics of chiral properties of protein secondary structure elements. Specifically, a closed right-handed alpha-helix of more than six residues long is shown formally to be non-planar and has the L topology. Antiparallel beta-sheets are planar. Two parallel beta-strands each of at least three residues in length, however, constitute a non-planar structural element and can have either L or D topology. The favored right-handed crossover for parallel beta-sheets has the L form, the same as the right-handed alpha-helix. This topological description of the hydrogen bonding in secondary structures may be extended to higher levels of protein structure and may provide a conceptual framework for studying complex protein architecture in general.

Chemical Phenomena↗
PubMed1999

Molecular configuration of Rh D epitopes as defined by site-directed mutagenesis and expression of mutant Rh constructs in K562 erythroleukemia cells.

The Rh D antigen is the most clinically important protein blood group antigen of the erythrocyte. It is expressed as a collection of at least 37 different epitopes. The external domains of the Rh D protein involved in epitope presentation have been predicted based on the analysis of variant Rh D protein structures inferred from their cDNA sequences and their D epitope expression. This analysis can never be absolute because (1) most partial D phenotypes involve multiple amino acid changes in the Rh D protein and (2) deficiency for 1 or more epitopes may be due to gross structural alteration in the variant Rh D protein structure. We report here the amino acid requirements for the majority of D epitopes. They have been defined by generating a series of novel Rh mutant constructs by mutagenesis using an Rh cE cDNA as template and mutagenic oligonucleotide primers. When transfected into K562 cells, the D epitope expression of the derived mutant clones was then assessed by flow cytometry. The introduction of 9 externally predicted Rh D-specific amino acids on the Rh cE protein was sufficient to express 80% of all tested D epitopes, whereas other clones expressed none. We concluded from our data that the D epitope expression is consistent with at least 6 different epitope clusters localized on external regions of the Rh D protein, most involving overlapping regions within external loops 3, 4, and 6.

Epitopes↗
PubMed2003

Nucleation-dependent conformational conversion of the Y145Stop variant of human prion protein: structural clues for prion propagation.

One of the most intriguing disease-related mutations in human prion protein (PrP) is the Tyr to Stop codon substitution at position 145. This mutation results in a Gerstmann-Straussler-Scheinker-like disease with extensive PrP amyloid deposits in the brain. Here, we provide evidence for a spontaneous conversion of the recombinant polypeptide corresponding to the Y145Stop variant (huPrP23-144) from a monomeric unordered state to a fibrillar form. This conversion is characterized by a protein concentration-dependent lag phase and has characteristics of a nucleation-dependent polymerization. Atomic force microscopy shows that huPrP23-144 fibrils are characterized by an apparent periodicity along the long axis, with an average period of 20 nm. Fourier-transform infrared spectra indicate that the conversion is associated with formation of beta-sheet structure. However, the infrared bands for huPrP23-144 are quite different from those for a synthetic peptide PrP106-126, suggesting conformational non-equivalence of beta-structures in the disease-associated Y145Stop variant and a frequently used short model peptide. To identify the region that is critical for the self-seeded assembly of huPrP23-144 amyloid, experiments were performed by using the recombinant polypeptides corresponding to prion protein fragments 23-114, 23-124, 23-134, 23-137, 23-139, and 23-141. Importantly, none of the fragments ending before residue 139 showed a propensity for conformational conversion to amyloid fibrils, indicating that residues within the 138-141 region are essential for this conversion.

Amino Acid Substitution↗
PubMed1991

Proton nuclear magnetic resonance as a probe of differences in structure between the C102T and F82S,C102T variants of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae.

Differences in chemical shifts and in nuclear Overhauser effects between the C102T and F82S,C102T variants of Saccharomyces cerevisiae iso-1-cytochrome c in both the reduced and oxidized forms are reported and analyzed. There is evidence for small conformational differences in both oxidation states of the double variant near position 82. Differences in structure are more evident in the oxidized forms of the variants. These differences extend to distant parts of the protein. It is concluded that the oxidized double variant has undergone a small rearrangement of several regions of the protein that are linked by a hydrogen-bond network. It is shown that the rearrangement involves hydrogen bonds associated with the two heme propionates and associated water molecules. The deductions from nuclear magnetic resonance data are compared with the differences in the crystal structures of the reduced forms of wild-type protein and the F82S variant [Louie, G. V., Pielak, G. J., Smith, M., & Brayer, G. D. (1988) Biochemistry 27, 7870-7876].

Amino Acid Sequence↗
PubMed1998

Solution structure of an artificial Fe8S8 ferredoxin: the D13C variant of Bacillus schlegelii Fe7S8 ferredoxin.

The solution structure of the D13C variant of the thermostable Fe7S8 ferredoxin from Bacillus schlegelii has been determined by 1H-NMR spectroscopy in its oxidized form. In a variable-temperature NMR study the D13C variant was as thermostable (up to 90 degrees C) as the wild-type protein (WT). Seventy-five out of 77 amino acid residues and 81% of all theoretically expected proton resonances in the D13C Fe8S8 protein have been assigned. Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR-derived dihedral angle constraints for the cluster-ligating cysteines. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms. The overall folding of the WT is maintained in the mutant, except for the immediate vicinity of the new cysteine, which becomes much more similar to native Fe8S8 proteins. The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins. Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found. The two conformations are structurally indistinguishable. Temperature-dependent NMR experiments show that one of them is thermodynamically more stable than the other.

Amino Acid Sequence↗
PubMed2002

[Effect of laminin on karyotypic variability in cell line of Indian muntjac skin fibroblasts].

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblasts cell line M and karyotypic variant of this line M' on cultivation on a laminin 2/4 coated surface. In cell line M, cultivated on the laminin-coated surface for 4 and 14 days, and in karyotypic variant M', cultivated for 2, 4 and 14 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with model numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. As a result, new modal chromosome numbers form. The frequency of cells with 4 chromosomes increases significantly; as a rule, such cells are absent in the control cell variants. Many new additional structural variants of the karyotype (SVK) appear. Detachment of cells M' from the laminin-coated surface followed by a 2 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for chromosomal number. The frequency of chromosomal aberrations on cultivation of the laminin-coated surface does not change relatively to controls. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population.

Animals↗