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PubMed1997

Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein.

The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 A, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pKa1 = 4.9 +/- 0.1, pKa2 = 12.0 +/- 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A., Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact protein and a partially deuterated form strongly suggests that excited-state proton transfer is not coupled to fluorescence emission.

Animals↗
PubMedDate not supplied

Structural and functional properties of IL-4delta2, an alternative splice variant of human IL-4.

Structural and functional properties of recombinant IL-4delta2, a naturally occurring splice variant of human IL-4 with a deletion of the loop region 22-37, have been analyzed. IL-4delta2 has alpha-helical structure and most likely preserves the "up-up-down-down" topology typical of the four-helix-bundle cytokines. IL-4delta2 interacts specifically with the alpha chain of IL-4R and competes effectively with IL-4 for the common binding sites. Thus, IL-4delta2 may act as a regulator of the cytokine net, being the natural antagonist of IL-4.

Alternative Splicing↗
PubMed2024

Functional analysis of a novel nonsense PPP1R12A variant in a Chinese family with infantile epilepsy.

BACKGROUND: Defects in PPP1R12A can lead to genitourinary and/or brain malformation syndrome (GUBS). GUBS is primarily characterized by neurological or genitourinary system abnormalities, but a few reported cases are associated with neonatal seizures. Here, we report a case of a female newborn with neonatal seizures caused by a novel variant in PPP1R12A, aiming to enhance the clinical and variant data of genetic factors related to epilepsy in early life. METHODS: Whole-exome and Sanger sequencing were used for familial variant assessment, and bioinformatics was employed to annotate the variant. A structural model of the mutant protein was simulated using molecular dynamics (MD), and the free binding energy between PPP1R12A and PPP1CB was analyzed. A mutant plasmid was constructed, and mutant protein expression was analyzed using western blotting (WB), and the interaction between the mutant and PPP1CB proteins using co-immunoprecipitation (Co-IP) experiments. RESULTS: The patient experienced tonic-clonic seizures on the second day after birth. Genetic testing revealed a heterozygous variant in PPP1R12A, NM_002480.3:c.2533 C > T (p.Arg845Ter). Both parents had the wild-type gene. MD suggested that loss of the C-terminal structure in the mutant protein altered its structural stability and increased the binding energy with PPP1CB, indicating unstable protein-protein interactions. On WB, a low-molecular-weight band was observed, indicating that the protein was truncated. Co-IP indicated that the mutant protein no longer interacted with PPP1CB, indicating an effect on the structural stability of the myosin phase complex. CONCLUSION: The PPP1R12A c.2533 C > T variant may explain the neonatal seizures in the present case. The findings of this study expand the spectrum of PPP1R12A variants and highlight the potential significance of truncated proteins in the pathogenesis of GUBS.

Female↗
PubMedDate not supplied

Memory performance of young, middle, and old adults.

Young, middle-aged, and older adults were assessed on tasks operationally defining short-term memory structural limitations and process abilities. The tasks were typical of previously related memory research with children and adolescents: monaural and dichotic word lists of variant association structure and rate of presentation. Although a number of minor chronological age-related differences were noted between monaural and dichotic word list performances, the findings indicated both structural capacity and organizational strategy deficiencies over successive adult age groups. In addition, older adults indicated significantly more heterogeneity of performance variance, both between and within age group comparisons. Results were summarized within the Cattell-Horn developmental model of fluid and crystallized abilities.

Adult↗
PubMed1978

Fine structure of an unusual spongy variant of medulloblastoma.

The prominent finding on electron microscopy of an unusual spongy variant of medulloblastoma was the marked distention of a well-developed smooth endoplasmic reticulum of the tumor cells. To the authors' knowledge, this fine structural feature has not been described previously in this tumor.

Adult↗
PubMed1998

The crystal structure of amyloidogenic Leu55 --> Pro transthyretin variant reveals a possible pathway for transthyretin polymerization into amyloid fibrils.

The x-ray crystal structure of the amyloidogenic Leu55 --> Pro transthyretin (TTR) variant, implicated as the causative agent in early-onset familial amyloidotic polyneuropathy (Jacobson, D. R., McFarlin, D. E., Kane, I., and Buxbaum, J. N. (1992) Hum. Genet. 89, 353-356), has been solved by molecular replacement, refined at 2.7 A to a Rcryst value of 0.190 (Fobs > 2.0sigma), and compared with wild-type transthyretin to understand the molecular mechanism(s) involved in amyloidogenesis. Leu55 --> Pro TTR crystallizes in space group C2, with eight monomers in the asymmetric unit, and the observed packing contacts are considerably different from those described for the wild-type protein. Refinement of the crystal structure shows that the proline for leucine substitution disrupts the hydrogen bonds between strands D and A, resulting in different interface contacts. Based on the assumption that the observed packing contacts may be significant for amyloidogenesis, a model for the TTR amyloid is proposed. It consists of a tubular structure with inner and outer diameters approximately of 30 and 100 A and four monomers per cross-section.

Amino Acid Sequence↗
PubMed1980

Molecular structure of adeno-associated virus variant DNA.

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.

Adenoviruses, Human↗
PubMed1985

Structures of N-terminally acetylated proteins.

Primary structures of 250 characterized proteins with N-terminally acetylated residues were correlated with residue distributions and other data. Excluding multiple forms derived from characterized species variants, the structures represent 105 different types of acetylated proteins. Results of comparisons extend previous suggestions based on fewer structures and define relationships further. The N-terminal residue that is acetylated is of a limited type and is frequently a small residue, with a heavy over-representation of serine and alanine. However, the occurrence of methionine at the acetylated position is also high, whereas that of glycine is less frequent than previously estimated. Lysine is over-represented in the N-terminal region, as is aspartic and glutamic acids at a few positions close to the acetylated N-terminus (especially the adjacent position). Finally, distributions of branched-chain residues in the N-terminal region of acetylated proteins are altered in relation to those of proteins in general, isoleucine is over-represented, and leucine and valine are under-represented. The results suggest that alpha-amino-acetylated proteins have special residues in N-terminally non-hydrophobic structures. Data are compatible with a protective function for acetylation but do not exclude further role(s) in processing or other special functions.

Acetylation↗
PubMed1992

From molecular variant to disease: initial steps in evaluating the association of transthyretin M119 with disease.

Traditionally, clinical research has sought to determine the molecular basis of clinical signs and symptoms. Increasingly, the traditional process will be reversed, as many structural protein variants are elucidated as a result of powerful PCR-based methods. Herein we describe a variant of transthyretin (TTR) found by direct genomic sequencing and illustrate the utility of PASA (PCR amplification of specific alleles) in the initial characterization of such variants. TTR is an intriguing protein of unknown function, but deposition of mutant TTR produces familial amyloidotic polyneuropathy (FAP). We identify a carrier of a variant TTR in which threonine119 is changed to methionine (T119----M). T119 is invariant in five mammalian species, suggesting that this residue is important for normal protein function. To determine the frequency of the M119 variant, individuals of northern- and western-European descent were rapidly screened by generating a PASA assay for the sequence change. Four additional individuals were found to be heterozygous for the mutation, for a total of five M119 alleles in 1,666 genes (1/333). Clinical records, initial clinical interviews, and family history of these patients hint at a high frequency of early-onset venous insufficiency and perhaps mild renal dysfunction. Haplotype analysis on the heterozygotes could be performed, despite the absence of samples from relatives, by performing "double PASA." The haplotype data suggest that the M119 variant derives from a common ancestor. The putative functional deficiency caused by TTR M119 should be most marked in the homozygotes, who can be calculated to occur in 1/100,000 conceptions. If viable, these individuals may provide important clues about the physiological role of TTR. Although the nature (if any) of disease caused by TTR M119 remains to be defined, the genetic and clinical data indicate that this mutation does not cause FAP. Future family studies can determine whether the heterozygous state for TTR M119 cosegregates with a disease or trait.

Base Sequence↗
PubMed1991

Localization of extensive deletions in the structural genes of two neurotropic variants of murine coronavirus JHM.

The intracellular RNA of two neurotropic variants of the JHM strain of mouse hepatitis virus (MHV) independently isolated from the brain and spinal cord of an infected Wistar Furth rat were compared with that of the parental virus. The mRNAs corresponding to the genes encoding the peplomer (S) and the hemagglutinin-esterase (HE) proteins of the variant viruses were found to be smaller in size. The possible sequence changes were studied by oligonucleotide fingerprinting and direct RNA sequencing. Both variants have a large deletion of 246 amino acids in the carboxy-terminal end of the HE protein. However, this truncated protein was not detected in the infected cells, suggesting either a translational regulation or rapid degradation of the truncated protein in these cells. The variant virus isolated from the spinal cord has a second deletion of 147 amino acids in the amino-terminal half of the S protein. This deletion site corresponds to a hypervariable region where deletions have been frequently noted among MHV variants with different biological properties. These findings suggest that the changes in pathogenic properties of the two neural isolates are associated with drastic alterations of the viral structural glycoproteins.

Base Sequence↗
PubMed1991

Detection of a key tertiary interaction in the highly conserved GTPase center of large subunit ribosomal RNA.

Searches of ribosomal RNA sequences for compensatory base changes preserving Watson-Crick base pairing have led to detailed models of the conserved secondary structures of these RNAs. In principle, tertiary interactions can also be detected by searches for phylogenetically covariant bases. Within a highly conserved region of the large subunit ribosomal RNA termed the "GTPase center," the bases G-1056-U-1082.A-1086 are found in all eubacteria (Escherichia coli numbering), while A-1056.C-1082.G-1086 are found at the homologous positions in eukaryotes; archaebacteria fall into either category with some exceptions. Either sequence can potentially form a similar set of hydrogen bonds connecting the 3 bases. To determine the contribution of these 3 bases to RNA tertiary structure, sequence variants were made in RNA fragments covering the GTPase center. Correct folding of the RNA fragments was assayed by measuring the binding affinities of two different ligands that recognize the RNA tertiary structure: the highly conserved ribosomal protein L11, which is normally associated with the GTPase center RNA, and the peptide antibiotic thiostrepton, which inhibits the GTPase activity of eubacterial and some archaebacterial ribosomes. The results strongly support the existence of a base pair between positions 1082 and 1086: single mutations at either position weaken both L11 and thiostrepton binding by approximately 10-fold or more, while compensatory double mutations bind the ligands nearly as well as the wild-type E. coli sequence. Variants at position 1056 have little effect on either L11 or thiostrepton binding; a 3-base interaction is therefore not supported by these experiments. A base pair between positions 1082 and 1086 strongly constrains the geometry with which three helical segments join in the middle of the GTPase center.

Base Sequence↗
PubMed1989

Human erythrocyte glycophorin C. Gene structure and rearrangement in genetic variants.

We have previously shown that a deletion of approximately 3 kilobases in the unique glycophorin C (GPC) gene, which encodes for the human erythrocyte glycophorins C and D, is associated with the Gerbich (Ge) blood group deficiency (Ge-2,-3 and Ge-2,+3 types) (Le van Kim, C., Colin, Y., Blanchard, D., Dahr, W., London, J. & Cartron, J.P. (1987) Eur. J. Biochem. 165, 571-579). We have now isolated and characterized the structure of the GPC gene from the common Ge+2,+3 donors and from a Ge-2,-3 variant (Ge-2,-3 gene). The GPC gene is organized in four exons distributed over 13.5-kilobase pairs (kbp) DNA and contains two directly repeated domains of 3.4 kbp in length which are likely derived from the recent duplication of a unique ancestral domain. Restriction mapping and sequence analysis indicate that a 3.4-kbp deletion within this gene, arising probably by unequal crossing over between the two repeated domains, is responsible for the formation of the Ge-2,-3 gene. The breakpoints of the deletion are located within introns 2 and 3, and therefore exon 3 is removed. The defective gene is transcribed as a mRNA with a continuous open reading frame extending over 300 nucleotides which is translated into an unusual sialoglycoprotein present on Ge-2,-3 red cells. The primary structure of this new glycoprotein has been deduced from nucleotide sequencing. It is proposed in addition, that another 3.4-kb deletion within the GPC gene eliminates exon 2 only by a similar mechanism and generates a defective gene encoding for the abnormal glycoprotein present on Ge-2,+3 erythrocytes. Interestingly, the same deletion which lead to the rare Ge-2,-3 genetic condition, occurred spontaneously and frequently in the cloned GPC gene during the propagation of the recombinant phages in Escherichia coli. From these observations we suggest that the Ge-2,-3 and Ge-2,+3 genes might represent the two allelic forms of a unique ancestral form of the GPC gene, following successive internal duplication and deletion events.

Amino Acid Sequence↗
PubMed1999

Receptor binding activity and in vitro biological activity of the human FSH charge isoforms as disclosed by heterologous and homologous assay systems: implications for the structure-function relationship of the FSH variants.

Follicle-stimulating hormone (FSH) is produced and secreted in multiple molecular forms. These isoforms differ in their oligosaccharide structures, which determine the particular behavior of a given variant in in vitro and in vivo systems. Employing heterologous cell assay systems, this and other laboratories have shown that highly sialylated human FSH variants exhibit lower receptor binding/immunoactivity as well as in vitro bioactivity/immunoactivity relationships than their less sialylated counterparts. It is not known, however, whether this characteristic behavior of the FSH isoforms is reproduced by homologous assay systems, in which unique variants of the receptor are presumptively expressed. To gain further insights into the structure-activity relationship of the various FSH isoforms, we analyzed the capacity of nine charge isoforms obtained after high-resolution chromatofocusing (pH window, 7.10 to <3.80) of anterior pituitary glycoprotein extracts to bind and activate their cognate receptor expressed by naturally occurring heterologous cell systems (rat granulosa cells and seminiferous tubule homogenates) as well as by human embryonic kidney-derived 293 (HEK-293) cells transfected with the human FSH (FSH-R) receptor cDNA. In both (heterologous and homologous) receptor assay systems, the isoforms displaced 125I-labeled FSH from the receptor in a dose-response manner; however, whereas in the heterologous systems, the receptor binding activity varied according to the elution pH value/sialic content of the isoforms, with the less acidic variants exhibiting higher receptor binding activity (r = 0.851 and 0.495 [p < 0.01 and p < 0.05] for the granulosa cell and testicular homogenate receptor assay systems, respectively) than the more acidic/sialylated analogs, in the homologous assay, this relationship was practically absent (r = 0.372, p N.S.). The capacity of the isoforms to induce androgen aromatization by rat granulosa cells followed the same trend shown by its corresponding receptor assay system (r = 0.864, p < 0.01). Interestingly and in contrast to the results observed in the homologous receptor binding assay, the ability of the isoforms to induce cAMP production by HEK-293 cells varied according to their elution pH value, with the more sialylated isoforms exhibiting lower potency than their less acidic counterparts (r = 0.852, p < 0.01). The results yielded by the heterologous assays suggest that the different potency of the isoforms to elicit a biological effect in a naturally occurring receptor system depends primarily on the particular affinity of the receptor molecule for each isoform. The existence of a clear dissociation between receptor binding and signal transduction in the homologous system indicate that this later function is rather related to the different ability of the FSH glycosylation variants to induce and/or stabilize distinct receptor conformations that may permit preferential or different degrees of activation/inhibition of a given signal transduction pathway. Thus, the human FSH receptor-transducer system apparently possesses sufficient versatility to respond in a different manner to glycosylation-dependent diverse FSH signals.

Animals↗
PubMed2025

Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumor-normal pair.

The Genome in a Bottle Consortium (GIAB), hosted by the National Institute of Standards and Technology (NIST), is developing new matched tumor-normal samples, the first explicitly consented for public dissemination of genomic data and cell lines. Here, we describe a comprehensive genomic dataset from the first individual, HG008, including DNA from an adherent, epithelial-like pancreatic ductal adenocarcinoma (PDAC) tumor cell line and matched normal cells from duodenal and pancreatic tissues. Data for the tumor-normal matched samples comes from seventeen distinct state-of-the-art whole genome measurement technologies, including high depth short and long-read bulk whole genome sequencing (WGS), single cell WGS, Hi-C, and karyotyping. These data will be used by the GIAB Consortium to develop matched tumor-normal benchmarks for somatic variant detection. We expect these data to facilitate innovation for whole genome measurement technologies, de novo assembly of tumor and normal genomes, and bioinformatic tools to identify small and structural somatic variants. This first-of-its-kind broadly consented open-access resource will facilitate further understanding of sequencing methods used for cancer biology.

Humans↗
PubMed2001

Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity.

SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of alpha-helical and beta-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of alpha-helical and beta-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the alpha-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion.

Amino Acid Sequence↗
PubMed1998

Oxidation of guanines in the iron-responsive element RNA: similar structures from chemical modification and recent NMR studies.

BACKGROUND: The translation or stability of the mRNAs from ferritin, maconitase, erythroid aminoevulinate synthase and the transferrin receptor is controlled by the binding of two iron regulatory proteins to a family of hairpin-forming RNA sequences called iron-responsive elements (IREs). The determination of high-resolution nuclear magnetic resonance (NMR) structures of IRE variants suggests an unusual hexaloop structure, leading to an intra-loop G-C base pair and a highly exposed loop guanine, and a special internal loop/bulge in the ferritin IRE involving a shift in base pairing not predicted with standard algorithms. RESULTS: Cleavage of synthetic 55- and 30-mer RNA oligonucleotides corresponding to the ferritin IRE with complexes based on oxoruthenium(IV) shows enhanced reactivity at a hexaloop guanine and at a guanine adjacent to the internal loop/bulge with strong protection at a guanine in the internal loop/bulge. These results are consistent with the recent NMR structures. The synthetic 55-mer RNA binds the iron-regulatory protein from rabbit reticulocyte lysates. The DNA analogs of the 55- and 30-mers do not show the same reactivity pattern. CONCLUSIONS: The chemical reactivity of the guanines in the ferritin IRE towards oxoruthenium(IV) supports the published NMR structures and the known oxidation chemistry of the metal complexes. The results constitute progress towards developing stand-alone chemical nucleases that reveal significant structural properties and provide results that can ultimately be used to constrain molecular modeling.

Animals↗
PubMed2026

Somatic mosaicism in the brain: linking development, ageing and neurodegeneration.

Somatic mosaicism is increasingly recognized as a pervasive feature of the human brain and a potential contributor to neurological disease across the lifespan. Unlike germline variants, somatic variants arise post-zygotically and are unevenly distributed across regions, cell types and even individual neurons, enabling focal biological effects that can scale to network-level dysfunction. In this Review, we synthesize current evidence that developmental timing, clonal architecture and cell-type-specific selective pressures shape how somatic variants influence brain structure and function. Early embryonic variants can produce broad regional clones and severe phenotypes, whereas later events are usually more restricted; with ageing, ongoing DNA damage and imperfect repair generate private variants that might cumulatively reduce cellular resilience. We also summarize advances in detection approaches, including bulk, error-corrected and single-cell sequencing, and discuss their strengths and current limitations for clinical translation. Emerging data link brain somatic variants to neurodevelopmental and neurodegenerative phenotypes, supporting a unified framework in which mosaic genetics bridges focal lesions and distributed neurological syndromes. Integrating genomic, cellular and physiological analyses in longitudinal human studies will be essential to define causality, identify biomarkers and guide future targeted interventions.

Journal Article↗
PubMed1980

Three-dimensional structure of a protein from scorpion venom: a new structural class of neurotoxins.

The three-dimensional crystal structure of variant-3 toxin from the scorpion Centruroides sculpturatus Ewing has been determined at 3 A resolution. Phases were obtained by use of K2PtCl4 and K2IrCl6 derivatives. The most prominent secondary structural features are two and a half turns of alpha-helix and a three-strand stretch of antiparallel beta-sheet, which runs parallel to the alpha-helix. The helix is connected to the middle strand of the beta-sheet by two disulfide bridges; a third disulfide bridge is located nearby. Several loops extend out of this dense core of secondary structure. The largest loop is joined to the COOH terminus of the molecule by the fourth disulfide bridge. The overall shape of the molecule resembles a right-hand fist: the alpha-helix runs along the knuckles of the fist; the beta-sheet lies along the second and third joints of the fingers; the thumb is defined by two short loops that are composed of residues 16-21 and residues 41-46; the wrist corresponds to the COOH-terminal stretch of residues 52-65 and a loop composed of residues 5-14; and the second joint of the little finger is near the NH2 terminus of the molecule. The alpha-carbon backbone displays a large flat surface that lies along the second joints of the fingers and the heel of the hand in the fist model. Several of the conserved residues in the scorpion neurotoxins are clustered on this surface, which may play a role in interactions of scorpion toxins with sodium channels of excitable membranes.

Hydrogen Bonding↗