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PubMed1988

Structural analysis of recombinant soluble human interleukin-2 receptor. Primary structure, assignment of disulfide bonds and core IL-2 binding structure.

A purified soluble and functional form of recombinant human interleukin-2 receptor, engineered and expressed in Chinese hamster ovary cells, was structurally characterized. The primary sequence of this 224 amino acid recombinant protein which lacks most of the carboxy-terminal transmembrane and cytoplasmic portions of the intact protein was established by sequence analyses. The disulfide bonds were assigned by comparative peptide mapping of the reduced and non-reduced peptide digests. As in the case of natural interleukin-2 receptor they occur between cysteines 3-147, 46-104, 131-163, and 28/30-59/61. Based on assignment of the disulfide bonds, a structural model of the interleukin-2 receptor for interleukin-2 binding is proposed.

Amino Acid Sequence↗
PubMed2001

[Structural analysis for psychosocial factors including health locus of control (HLC) and sense of coherence (SOC) associated with lifestyle-related diseases].

The purpose of this study was to clarify psychosocial characteristics associated with preventive health behavior for lifestyle-related diseases. The author performed objective health examinations and gave questionnaires to 289 men (39.7 +/- 11.8 years, mean +/- SD) and 80 women (32.8 +/- 10.4 years) engaged in office work. Psychosocial factors included lifestyle and perceived stress, as well as the health locus of control (HLC) and sense of coherence (SOC) as newly developed indicators for health behavior. The principal component analysis for men did not extract lifestyle from the psychosocial structures. Multiple regression analysis showed that internal HLC (IHLC), chance HLC (CHLC), professional HLC (PHLC) and stress significantly contributed to SOC. Principal component analysis using psychosocial factors in women showed two psychosocial structures, i.e. the second principal (high SOC, high lifestyle, and low stress) and the 4th principal components (high supernatural HLC, and high PHLC). Both components were negatively correlated with systolic blood pressure. SOC was recognized to be negatively associated with age, stress, and total cholesterol, and positively with IHLC, FHLC, lifestyle, and gamma-GTP using multiple regression analysis for women. These results indicated a distinguishable sex difference regarding the involvement of psychosocial factors including HLC and SOC in objective health. SOC seems likely to be involved not in objective health, but closely with stress, suggesting a direct influence on mental health. Lifestyle should be divided into more detailed categories such as smoking and salt intake. Structural analysis of women suggests that SOC is involved directly or indirectly through lifestyle in objective health, different from men. To further clarify causal relationships between psychosocial factors and risk factors for lifestyle-related diseases, a longitudinal study is necessary based on these results.

Adult↗
PubMed1992

Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis.

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.

Affinity Labels↗
PubMed1989

[Functional and structural analysis of the Lyb-2 system].

The Lyb-2 system of the mouse is a B cell-specific cell surface molecule encoded by a gene on chromosome 4. We have previously reported that Lyb-2 is involved in an early phase of B cell differentiation, specifically a process mediated by B cell stimulatory factor-1 (BSF-1) or-interleukin 4. It is thus very important to define the functional role and structural features of Lyb-2 molecule for the understanding of the regulatory mechanisms of B cell activation initiated by BSF-1. In our attempt to resolve these issues, we found that Lyb-2 antibody inhibits activation processes mediated by BSF-1 such as induction of Ia antigen on the B cell surface and of IgG1 production in lipopolysaccharide-activated B cells, suggesting that Lyb-2 molecule participates in a signaling event triggered by the binding of BSF-1 to its receptor. In the structural analysis, we found that in contrast to previous reports, Lyb-2 is not a monomer of 45 kilodaltons (kDa) but is composed of two components with molecular weight of 45 kDa and 105 kDa. Reduced and nonreduced two-dimensional electrophoresis analysis further revealed that Lyb-2 molecules are present on the B cell surface in two forms, a disulfide-bonded heterodimer of 45 kDa and 105 kDa chains and a 45 kDa homatrimer. How the structural uniqueness of Lyb-2 is related to its functional expression remains to be determined.

Animals↗
PubMed2002

Activator recruitment by the general transcription machinery: X-ray structural analysis of the Oct-1 POU domain/human U1 octamer/SNAP190 peptide ternary complex.

Transcriptional activation of the human U1 snRNA genes is dependent on a noncanonical octamer element contained within an upstream enhancer. The U1 octamer only weakly recruits the Oct-1 POU domain, although recruitment is stimulated by a peptide containing the Oct-1-binding domain of SNAP190. Structural analysis of the Oct-1 POU domain/U1 octamer/SNAP190 peptide complex revealed that SNAP190 makes extensive protein contacts with the Oct-1 POU-specific domain and with the DNA phosphate backbone within the enhancer. Although SNAP190 and OCA-B both interact with the Oct-1 POU domain through the same Oct-1 interface, a single nucleotide within the U1 octamer ablates OCA-B recruitment without compromising activator recruitment by SNAP190.

DNA-Binding Proteins↗
PubMed1989

Observed family interactions among subtypes of eating disorders using structural analysis of social behavior.

Compared observations of family interactions among anorexic, bulimic-anorexic, bulimic, and normal families. A total of 74 family triads participated, including father, mother, and teenage daughter. Each family was videotaped during a 10-min discussion of the daughter's separation from the family. These tapes were coded using Benjamin's structural analysis of social behavior (SASB) model and observational schema. The results showed that the SASB methodology differentiated clinical from normal families and that there were unique patterns among subtypes of eating disorders. Specifically, parents of anorexics communicated a double message of nurturant affection combined with neglect of their daughter's needs to express themselves and their feelings. Anorexic daughters, in turn, were ambivalent about disclosing their feelings versus submitting to their parents. In contrast, bulimics and their parents were hostilely enmeshed and, for them, this appeared to undermine the daughter's separation and self-assertion. These findings are consistent with current theory and research on anorexia and bulimia.

Adolescent↗
PubMed1995

Structural analysis of social behavior and perceptions of caregiving.

The stress and burden of caregiving have been well established as correlates of changes in the relationship with a person needing care, but there has been less research on the association of caregiving with psychosocial variables, especially relationship patterns. In this article, these issues were addressed using Structural Analysis of Social Behavior (SASB; Benjamin, 1974, 1984, 1993), a self-report measure of perceptions of interpersonal relationships and self-concept, and the Symptom Check List-90--Revised (SCL-90-R; Derogatis, 1983), a self-report measure of distress and symptoms. SASB variables measuring attachment, interdependence, and interpersonal/intrapsychic conflict successfully differentiated community-active caregivers from noncaregivers. SASB measures provided substantially greater discrimination and classification accuracy than SCL-90-R variables.

Aged↗
PubMed1998

[Job satisfaction of sales people: a covariance structure analysis of the motivational process].

The purpose of this study was to investigate the relationship among job satisfaction, job involvement, and work motivation. Two hundred thirty-nine sales people completed a questionnaire of job satisfaction (pay, interpersonal relationship, work environment, and job content), job involvement, and work motivation. The data were analyzed with covariance structure analysis, and the model, which was constructed beforehand, fitted well with relatively high GFI and AGFI. Results of the analysis showed that job satisfaction, in terms of pay and interpersonal relationship, influenced job content satisfaction, which in turn indirectly influenced work motivation, mediated through job involvement. In addition, the data indicated that satisfaction with customer relationship was strongly related to job content satisfaction in the sample of sales people.

Adult↗
PubMed1999

Structural analysis of the thyroid hormone receptor ligand binding domain: studies using a quadrupole time-of-flight tandem mass spectrometer.

The overall architecture of the ligand binding domain (LBD) of members of the nuclear receptor superfamily are similar. There are now standard procedures to express and purify these proteins. A rapid and sensitive method for the structural analysis of these proteins is nano-electrospray tandem mass spectrometry. In the present study we have analysed the LBD of the human thyroid hormone receptor-beta-1 (TR-beta) by quadrupole time-of-flight tandem mass spectrometry. The intact protein was analysed in a carboxymethylated form in an attempt to identify which cysteine residues are located on the surface. The protein molecular weight (31 652.5 Da) was determined with an accuracy of +/-1 Da, while masses of tryptic fragments were determined with an accuracy of at least 75 ppm. The sequence coverage of the tryptic peptide mass map was 93.2 %. Tryptic peptides were subjected to collision-induced dissociation (CID) and the resulting product ions were mass measured with an accuracy of about 100 ppm. When accurate mass measurements were made with internal calibration, mass accuracies were improved to +/-2 ppm in mass spectra, and +/-20 ppm in CID spectra. From these data it was possible to determine the presence of post-translational modifications, locate the sites of carboxymethylation and, in addition, confirm the amino acid sequence of the expressed protein. To the best of our knowledge, this is the first characterisation of the TR-LBD-beta at the protein level.

Amino Acid Sequence↗
PubMed1998

alpha-Thalassaemia due to a single codon deletion in the alpha1-globin gene. Computational structural analysis of the new alpha-chain variant. Mutations in brief no. 132. Online.

A new unstable alpha-globin chain associated with alpha-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the alpha-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the alpha-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 and DK61 results in a structurally abnormal alpha-globin chain, not previously described, named Hb Clinic, which leads to the alpha-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this alpha-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer.

Codon↗
PubMed1992

Crystallization and preliminary X-ray structure analysis of pigeon egg-white lysozyme.

Calcium binding lysozyme from pigeon egg-white was crystallized by the hanging drop vapor diffusion technique using ammonium sulphate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), and have unit cell dimensions of a = 34.2 A, b = 34.8 A, and c = 99.4 A. One asymmetric unit contains one molecule of the pigeon lysozyme. The crystals diffract X-rays at least to 2.0 A resolution and are suitable for high resolution structure analysis. The diffraction data up to 3.0 A resolution were collected with a diffraction image processor, DIP100, using a Fuji imaging plate as an area detector. The structure was solved by the molecular replacement technique and refined to an R factor of 0.216. Least-squares fitting of the main-chains of pigeon egg-white lysozyme with those of chicken egg-white lysozyme and baboon alpha-lactalbumin showed that the main-chain folding of pigeon lysozyme is more similar to that of chicken lysozyme than that of alpha-lactalbumin. The largest differences between the pigeon and chicken lysozymes are in the surface loop regions.

Amino Acid Sequence↗
PubMed1997

Structural analysis of the oligosaccharide-alditols released by reductive beta-elimination from oviducal mucins of Rana temporaria.

The carbohydrate chains of the mucins which constitute the jelly coat surrounding the eggs of Rana temporaria were released by alkaline borohydride treatment. Neutral and acidic oligosaccharide-alditols were purified by ion-exchange chromatography and HPLC. From the structural analysis, based upon 1H and 13C-NMR spectroscopy in combination with MALDI-TOF, the following glycan units are proposed.

Animals↗
PubMed1984

Hand Test and the High School Personality Questionnaire: structural analysis.

The Hand Test and the High School Personality Questionnaire were administered to 15 boys and 15 girls in Grades 9 and 10 transferred to an alternative school for acting-out behavior. They were of average intelligence. Several significant correlations between Hand Test and High School Personality Questionnaire variables were noted. Results are discussed in reference to Structural Analysis.

Acting Out↗
PubMed1992

Crystallization and preliminary structural analysis of the replication terminator protein of Bacillus subtilis.

The replication terminator protein (RTP) is a dimeric molecule that binds specific sequences within the replication terminus of the Bacillus subtilis chromosome and prevents the passage of replication forks. The gene for RTP has been expressed in Escherichia coli, and the protein has been purified in amounts sufficient for structural studies by nuclear magnetic resonance (NMR) and x-ray crystallography. One-dimensional NMR experiments show that the protein has a well-folded compact tertiary structure, as well as a high alpha-helical content. Circular dichroism (CD) studies confirm this finding and show that approximately 32% of the protein is alpha-helical. The terminator protein has been crystallized as monoclinic plates that diffract to better than 2.5 A and are suitable for high resolution structural analysis. Precession photographs show the space group to be C2 with unit cell dimensions a = 77 A, b = 53 A, c = 70 A, and beta = 90 degrees, and two molecules occupy the asymmetric unit. With a view to producing crystals of an RTP.DNA complex, gel-shift assays were performed to establish the shortest sequence of DNA that is required for tight binding to RTP. These clearly show that two turns of DNA are required, centered on an 8-base pair consensus sequence, to elicit relatively stable binding.

Autoradiography↗
PubMed1986

Structural analysis of papaya polysaccharide II.

The structure of papaya polysaccharide II (PP II) isolated by Chandrasekaran et al. [Carbohydr. Res., 60 (1978) 105-115] has been investigated by methylation analysis of the carboxyl-reduced polymer and by partial hydrolysis of both the intact (arabinose, 31.0; rhamnose, 13.3; galactose, 42.6; glucuronic acid, 10.3; and 4-O-methylglucuronic acid, 2.8%), and carboxyl-reduced polymers. Methylation analysis of carboxyl-reduced PP II indicated a very highly branched structure in which approximately 39% of the galactopyranose units are disubstituted, 24% are monosubstituted, 20% are trisubstituted, and 17% are nonreducing end units. Methylation analysis of products of partial hydrolysis of both intact and carboxyl-reduced polymers indicated that the backbone of the polysaccharide is made up of galactosyl residues substituted at either O-3 or -6, that the principal aldobiouronic acid fragment is 6-O-(glucopyranosyluronic acid)galactose, that the rhamnosyl units are substituted at O-3 with either terminal arabinofuranosyl or galactopyranosyl groups, and that the rhamnosyl residues are themselves linked to glucuronic acid residues through O-4. From this information, a possible statistical fragment with six arabinofuranose and two galactopyranose nonreducing end-groups per 19 sugar units [five units in the main chain of (1----3)-linked galactopyranose units] is proposed.

Carbohydrate Conformation↗
PubMed1999

Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling.

Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM), a membrane receptor present on the surface of endothelial cells. The understanding of this complex biological system is in part limited due to the lack of integration of experimental and structural data. In the work presented here, we analyze the PC-T-TM pathway in the context of both types of information. First, structural analysis of the serine protease domain of PC suggests that a positively charged cluster of amino acids could be involved in the activation process. To investigate the importance of these basic amino acids, two recombinant PC mutants were constructed using computer-guided site-directed mutagenesis. The double mutant had the K62[217]N/K63[218]D substitution and in the single mutant, K86[241] was changed to S. Both mutants were activated by free thrombin at rates equivalent to that of wild-type PC (wt-PC) and they demonstrated similar calcium-dependent inhibition of their activation. The K86[241]S mutant and wt-PC were activated by thrombin bound to soluble TM at a similar rate. In contrast, the K62[217]N/ K63[218]D mutant was activated by the T-TM complex at a 10-fold lower catalytic efficiency due to a lowering in k(cat) and increase in Km. Molecular models for PC and thrombin bound to a segment of TM were developed. The experimental results and the modeling data both indicate that electrostatic interactions are of crucial importance to orient PC onto the T-TM complex. A key electropositive region centered around loops 37[191] and 60[214] of PC is defined. PC loop 37[191] is located 7-8 A from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 A away from TM EGF4. Both loops are far from thrombin. A key function of TM could be to create an additional binding site for PC. The Gla domain of PC points toward the membrane and away from thrombin or the EGF modules of TM during the activation process.

Cell Line↗
PubMed1991

Structural analysis of the N-linked oligosaccharides from murine glycophorin.

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.

Animals↗