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T Yuan

Publications and source records attributed to T Yuan.

At least 37 records · Page 2Linked to original sources
PubMed1998

Calcium-calmodulin-induced dimerization of the carboxyl-terminal domain from petunia glutamate decarboxylase. A novel calmodulin-peptide interaction motif.

The acidic, bilobed protein calmodulin (CaM; molecular mass of 16.7 kDa) can activate some 40 distinct proteins in a calcium-dependent manner. The majority of the CaM-binding domain regions of the target proteins are basic and hydrophobic in nature, are devoid of multiple negatively charged residues, and have a propensity to form an alpha-helix. The CaM-binding domain in the C-terminal region of petunia glutamate decarboxylase (PGD) is atypical because it contains five negatively charged residues. Therefore, we chose to study the binding of calcium-CaM to a 26-residue synthetic peptide encompassing the C-terminal region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titration studies showed that a single unique complex of calcium-CaM with two PGD peptides is formed. The formation of a 1:2 protein-peptide complex is unusual; normally, calcium-CaM forms 1:1 complexes with the majority of its target proteins. Circular dichroism spectroscopy showed that the bound PGD peptides have an alpha-helical structure. NMR studies of biosynthetically [methyl-13C]methionine-labeled CaM revealed that all the Met side chains in CaM are involved in the binding of the PGD peptides. Analysis of fluorescence spectra showed that the single Trp residue of the two peptides becomes bound to the N- and C-terminal lobes of CaM. These results predict that binding of calcium-CaM to PGD will give rise to dimerization of the protein, which may be necessary for activation. Possible models for the structure of the protein-peptide complex, such as a dimeric peptide structure, are discussed.

Amino Acid Sequence↗
PubMed1998

Characterization of the Ca2+ -dependent and -independent interactions between calmodulin and its binding domain of inducible nitric oxide synthase.

Most interactions of calmodulin (CaM) with its target proteins are Ca2+-dependent, but a few Ca2+-independent CaM-target protein interactions have been identified. One example is the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We describe here the characterization of the Ca2+-independent interaction between CaM and a synthetic peptide corresponding to the CaM-binding domain of murine macrophage iNOS using circular dichroism (CD) spectroscopy. The CD spectrum of free iNOS peptide indicated a beta-sheet conformation. The interaction of iNOS peptide with apo-CaM in the absence of Ca2+ resulted in the peptide acquiring a type II beta-turn structure. This is in contrast to the situation in the presence of Ca2+ in which case the peptide acquired an alpha-helical conformation upon interaction with CaM, i.e. similar to the Ca2+-dependent interactions of CaM with numerous targets such as myosin light chain kinase (MLCK). Consistent with this similar structural change, iNOS peptide inhibited the Ca2+-CaM-dependent activation of smooth muscle MLCK by competing with MLCK for binding to Ca2+-CaM. The Kd of Ca2+-CaM for iNOS peptide was calculated from competition assays to be 0.3 nM. These results indicate that the structure of the CaM-binding domain of iNOS is quite different when bound to apo-CaM than Ca2+-CaM.

Amino Acid Sequence↗
PubMed1998

Tryptophan fluorescence quenching by methionine and selenomethionine residues of calmodulin: orientation of peptide and protein binding.

The two interaction surfaces of the dumbbell-shaped calcium-regulatory protein calmodulin (CaM) are rich in the amino acid Met. In this work we have used fluorescence spectroscopy to study the role of these Met residues in binding the single Trp residue that is found in many CaM-binding domain peptides. This approach is facilitated by the absence of Trp residues in CaM. In addition to the wild-type protein, we studied CaM containing the unnatural amino acid selenomethionine (SeMet), which was biosynthetically substituted for its nine Met residues. Furthermore, a CaM mutant protein in which all four Met residues in the C-terminal domain were mutated to Leu, and the N-terminal domain contained either Met or the unnatural SeMet, was studied. The Trp fluorescence quantum yield of many Trp-containing CaM-binding peptides increases upon binding to calcium-CaM. Moreover, the emission wavelength of the Trp fluorescence is blue-shifted from 353 to 325-333 nm. These parameters indicate movement of Trp from a solvent exposed to a hydrophobic environment. The fluorescence results obtained with these four CaM variants showed that Se is very effective at quenching Trp fluorescence in the calmodulin-bound peptides from myosin light chain kinase (MLCK) and CaM kinase I, while S is somewhat effective (Se > S > C). The quenching effect is markedly distance dependent, as it only influences the Trp residue of the bound peptide (<=7 A) but has little effect on the two Tyr residues in the C-terminal domain of CaM (>=10 A). Since the Trp fluorescence quenching is very dramatic, the protein containing Leu's in the C-terminal domain and SeMet's in the N-terminal domain allowed us to directly determine the orientation of the MLCK and CaM kinase I peptides bound to CaM; in both cases the Trp residue binds to the C-terminal domain of CaM. Our data indicate that SeMet quenching of Trp fluorescence could become a simple and useful tool for studies of protein folding, and protein-protein and protein-peptide interactions.

Amino Acid Sequence↗
PubMed1998

Molecular mechanisms of calmodulin's functional versatility.

Calmodulin (CaM) is a primary Ca2+-binding protein found in all eukaryotic cells. It couples the intracellular Ca2+ signal to many essential cellular events by binding and regulating the activities of more than 40 different proteins and enzymes in a Ca2+-dependent manner. CaM contains two structurally similar domains connected by a flexible central linker. Each domain of the protein binds two Ca2+ ions with positive cooperativity. The binding of Ca2+ transforms the protein into its active form through a reorientation of the existing helices of the protein. The two helices in each helix-loop-helix Ca2+-binding motif are almost antiparallel in Ca2+-free CaM. The binding of Ca2+ induces concerted helical pair movements and changes the two helices in each Ca2+ binding motif to a nearly perpendicular orientation. These concerted helix pair movements are accompanied by dramatic changes on the molecular surface of the protein. Rather than exhibiting a flat, hydrophilic molecular surface as seen in Ca2+-free CaM, the Ca2+-saturated form of the protein contains a Met-rich, cavity-containing hydrophobic surface in each domain. These hydrophobic surfaces are largely responsible for the binding of CaM to its targets. The unique flexibility and high polarizability of the Met residues located at the entrance of each hydrophobic pocket together with other hydrophobic amino acid residues create adjustable, sticky interaction surface areas that can accommodate CaM's targets, which have various sizes and shapes. Therefore, CaM is able to bind to a large array of targets without obvious sequence homology. Upon binding to its target peptides, the unwinding of the central linker allows the two domains of the protein to engulf the hydrophobic face of target peptides of differing lengths. The binding of Ca2+ reduces the backbone flexibility of CaM. Formation of complexes with its target peptides further decreases the backbone motion of CaM.

Allosteric Regulation↗
PubMed1998

[The use of predforte in the transnasal endoscopic sinus surgery].

For finding a way to advance the healing of the operative cavity after transnasal endoscopic sinus surgery (TESS). Predforte (mixt 10% prednisolone acetate) was administered transnasally to the operative cavity of 75 cases who accepted TESS. 13 cases (17.5%) were brought about a striking effect, the good results were achieved on the 56 cases. The total effective rate was 92.0%. The results suggest that the mixture could be distributed better and effected longer in comparison with the solution of predinisolone acetate.

Adolescent↗
PubMed1997

NMR studies of caldesmon-calmodulin interactions.

The binding of the calcium-regulatory protein calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. Two regions of caldesmon have been identified as putative calmodulin-binding domains. We have earlier reported on the binding of one of these domains to calmodulin (Zhang & Vogel (1994) Biochemistry 33, 1163-1171). Here we have studied the binding of CaM to synthetic peptides of CaD which contain: (1) both the first and second CaM-binding domains; (2) the second CaM-binding domain; and (3) the sequence between the first and second CaM-binding domains. Two-dimensional transferred nuclear Overhauser enhancement proton NMR measurements as well as circular dichroism studies of a 22-residue peptide NKETAGLKVGVSSRINEWLTK, which contains the second CaM-binding domain, show that only the C-terminal half of the peptide becomes alpha-helical upon binding to CaM. Somewhat surprisingly, the shorter 9-residue peptide SRINEWLTK was sufficient to form a 1:1 complex with CaM; this peptide appears to bind as a 3(10)-helix. Proton-carbon-13 correlation NMR titration studies with specifically labeled [methyl-13C]methionine CaM were used to study the participation of the hydrophobic regions in both domains of the dumbbell shaped CaM in peptide binding. Binding of a 54-residue CaD peptide containing both CaM-binding domains affects all the 8 Met residues in the two hydrophobic domains of CaM (only Met 76 in the linker region of CaM is not involved), while binding of the second CaM-binding domain of CaD influences principally Met 51, 71, and Met 124, 144. Simultaneous binding to CaM of two peptides comprising the first and the second CaM-binding domains also caused changes to all Met residues except Met 76. Taken together, these data demonstrate that both CaM-binding domains of CaD can bind simultaneously to the two hydrophobic regions of CaM.

Amino Acid Sequence↗
PubMed1996

Endotoxin-induced cardiac depression is associated with decreased cardiac dihydropyridine receptors in rabbits.

Endotoxin depresses left ventricular (LV) contractility independently of alterations in loading conditions, acidosis, or hypoxia (Hung and Lew, 1993a). We evaluated if endotoxin-induced LV depression is associated with a decrease in functional L-type calcium channels, as reflected by the number of dihydropyridine receptors measured by [3H]-PN200-110 binding. New Zealand white rabbits were instrumented with sonomicrometers to measure the end-systolic pressure-volume relationship after i.v. saline (group 1, n = 6), 5 micrograms/kg endotoxin (group II, n = 6), or 10 micrograms/kg endotoxin (group III, n = 6). The end-systolic volume (ESV) measured at a matched end-systolic pressure did not change significantly over 6 h in group I (ESV changed by < 5 +/- 2% S.E.) and group II (ESV changed by < 3 +/- 2%), but increased markedly in group III (ESV increased 70 +/- 24%, P < 0.05), indicating LV systolic depression. We measured [3H]-PN200-110 binding in crude membrane homogenates from the left ventricle. There was a dose-dependent decrease in Bmax: 75 +/- 5 fmol/mg protein in group I, 62 +/- 3 fmol/mg in group II, and 56 +/- 5 fmol/mg in group III (P = 0.02 by ANOVA). Since the majority of dihydropyridine receptors are functional L-type calcium channels in rabbits (Lew et al., 1991), we conclude that a decreased number of dihydropyridine receptors contributes to endotoxin-induced LV depression.

Animals↗
PubMed1996

Comparative analysis of the amino- and carboxy-terminal domains of calmodulin by Fourier transform infrared spectroscopy.

Fourier transform infrared spectra were obtained for mammalian calmodulin and two of its fragments produced by limited proteolysis with trypsin TR1C (1-77) and TR2C (78-148). Experiments were done in H2O, D2O and D2O/trifluoroethanol (TFE) mixtures. Information about secondary structure was obtained from analysis of the amide I and II bands; while characteristic absorbances for tyrosine, phenylalanine and carboxylate groups were analyzed for changes in tertiary structure. Our data indicate that the secondary and tertiary structure is preserved in the two half molecules of CaM, both in the apo- and Ca(2+)-saturated state. Addition of the structure-inducing solvent TFE causes marked changes only in the apo-TR1C domain. The maximum wavenumber for the amide I band of the two domains of CaM in D2O was markedly different (1642 cm-1 for TR1C versus 1646/1648 cm-1 for Ca2+ and apo-TR2C). This renders the amide I band for the intact protein very broad in comparison to that in other proteins and is indicative of a distribution of alpha-helices with slightly different hydrogen bonding patterns.

Amino Acid Sequence↗
PubMed1995

Interaction of calmodulin with its binding domain of rat cerebellar nitric oxide synthase. A multinuclear NMR study.

The intercellular messenger nitric oxide is produced through the action of nitric oxide synthases, a class of enzymes that is regulated by calcium-calmodulin (CaM). In this work, the interaction of CaM with a 23-amino-acid residue synthetic peptide, encompassing the CaM-binding domain of constitutive rat cerebellar nitric oxide synthase (cNOS), was investigated by various NMR methods. Cadmium-113 NMR studies showed that binding of the cNOS peptide increased the affinity of CaM for metal ions and induced interdomain cooperativity in metal ion binding as earlier observed for complexes of CaM with myosin light chain kinase (MLCK) peptides. By using specific isotopically labeled [13C]methyl-Met and selenomethionine-substituted CaM in two-dimensional proton-detected 13C and 77Se NMR studies, we obtained evidence for the involvement of the Met residues of CaM in the binding of the cNOS peptide. These residues form two hydrophobic surface areas on CaM, and they are also involved in the binding of other target proteins. A nitroxide spin-labeled version of the cNOS peptide caused broadening only for NMR resonances in the N-terminal half of CaM, showing that the peptide binds with a C to N orientation to the N- and C-terminal domains of CaM. pH titration experiments of CaM dimethylated with [13C]formaldehyde show that Lys-75 (and Lys-148) experience a large increase in pKa upon peptide binding; this indicates an unraveling of part of the helical linker region of CaM upon cNOS peptide binding. Taken together, our data show that the cNOS and MLCK peptides bind in a closely analogous fashion to CaM.

Amino Acid Oxidoreductases↗
PubMed1995

Characterization of the calmodulin binding domain of SIV transmembrane glycoprotein by NMR and CD spectroscopy.

Recent experimental evidence has shown that the C-terminal peptide of the HIV/SIV transmembrane glycoprotein 41 (gp41) can bind very tightly to calmodulin (CaM). These findings imply a potential mechanism for HIV/SIV cytopathogenesis, which involves the uncoupling of some critical cellular signal transduction pathways that are normally mediated by CaM. Here, we present circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy studies of a 28-residue synthetic peptide, SIV-L, corresponding to the C-terminal portion of the SIV transmembrane glycoprotein gp41. CD studies recorded in aqueous solution show a dramatic increase in the amount of alpha-helical structure of the SIV-L peptide upon binding to calcium-CaM. Two-dimensional NMR experiments were performed to determine the secondary structure of the peptide in 25% aqueous trifluoroethanol solution. In this alpha-helix inducing solvent, the observed nuclear Overhauser effects, as well as the alpha 1H and alpha 13C chemical shift changes, demonstrate that a continuous alpha-helix is formed from W3 to L28, although there is some distortion around P17. This result is in accordance with those obtained for many other CaM-binding peptides. Subsequent one-dimensional NMR titration experiments of calcium-CaM and the SIV-L peptide suggest that the peptide can bind to CaM with a 1:1 stoichiometry and that the peptide binding involves both the N- and C-lobe of CaM. However, gel mobility shift assays suggest that the peptide CaM interaction may be more complicated, as oligomeric forms of CaM and the SIV-L peptide were found. These studies provide a potential molecular basis for HIV/SIV cytopathogenesis.

Amino Acid Sequence↗
PubMed1995

Serum amyloid A (SAA): influence on HDL-mediated cellular cholesterol efflux.

Normal high density lipoprotein (N-HDL) is remodeled during acute phase (AP) reactions by the association of serum amyloid A (SAA) and the depletion of apolipoprotein (apo) A-I. To determine the impact of this remodeling on HDL function, the capacities of N-HDL and AP-HDL to associate with and promote cholesterol efflux from human monocytic THP-1 cells were compared. THP-1 cells preferentially bound AP-HDL compared with N-HDL. Examination of the AP-HDL particles bound to THP-1 cells revealed a disproportionate association of an apoSAA-enriched, apoA-I-depleted subpopulation compared with the composition of the starting material. However, N-HDL and AP-HDL promoted cholesterol efflux from THP-1 cells equally efficiently and in a dose-dependent manner. When N-HDL was experimentally remodeled with apoSAA to achieve an apoprotein composition similar to that of the preferentially bound particles, cellular cholesterol efflux was reduced by 30%. The remodelling of HDL with apoSAA during the acute phase reaction alters cholesterol efflux only when apoSAA constitutes more than 50% of the HDL protein.

Apolipoprotein A-I↗
PubMed1995

Characterization of constitutive human serum amyloid A protein (SAA4) as an apolipoprotein.

Serum amyloid A proteins (SAAs), a family of homologous molecules, are apolipoproteins of high density lipoprotein (HDL). They can be divided into two groups. The first group comprises the well-characterized acute phase SAAs that associate with HDL during inflammation, thereby remodeling the HDL particle by displacing apolipoprotein (apo)A-I. The second group consists of the recently discovered constitutive SAAs, mouse SAA5 and human SAA4. They exist as minor apolipoproteins on HDL but constitute more than 90% of the total SAA during homeostasis. We have characterized human SAA4 as an apolipoprotein. During homeostasis, SAA4 is synthesized only in the liver. Purification of SAA4 has been described and its plasma concentration has been established at 55 +/- 13 micrograms/ml in 26 healthy individuals. It was present on all HDL density classes and very low density lipoprotein (VLDL) but was absent from low density lipoprotein (LDL). Using two-dimensional electrophoresis and phosphorimaging, SAA4 was found to be associated with a specific subpopulation of only three HDL particles, not involved in the initial cholesterol transfer from cells.

Acute-Phase Reaction↗
PubMed1994

Effects of increased rearing period body weights and early photostimulation on broiler breeder egg production.

Broiler breeder pullets were subjected to photostimulation (PS) at either 14, 17, or 20 wk of age after attaining greater than recommended BW during rearing in order to determine effects on age at onset of lay, egg weight, and egg production. During rearing, feed was consumed ad libitum (AL) or provided in allotments resulting in growth to projected BW of 2.8 (heavy, HV) or 2.3 kg (light, LT) at 20 wk. Rearing period feeding and age at PS treatments made up a 3 x 3 factorial arrangement. A single restricted feeding program was provided during lay. Although age at first egg in the AL treatment was earlier than in HV and LT treatments, total production by HV and LT birds was greater because peak and postpeak production by AL birds were depressed. Relative to PS at 20 wk, PS at 14 and 17 wk advanced age at first egg and increased cumulative production to 30 wk. However, early PS decreased peak production and, therefore, did not affect total production. Mean egg weight and settable egg production (egg weight > 50 g) were increased in HV and LT treatments. Egg weight and settable egg production were not affected by age at PS. These results indicate that the onset of lay by broiler breeders can be advanced by early PS and that increased BW facilitates this. However, allowing greater BW during rearing does not compensate for reduced early egg weights and results in decreased total production and mean egg weights when feed is provided at recommended levels during lay.

Age Factors↗
PubMed1993

A peptide analog of the calmodulin-binding domain of myosin light chain kinase adopts an alpha-helical structure in aqueous trifluoroethanol.

A 22-residue synthetic peptide encompassing the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase was studied by two-dimensional NMR and CD spectroscopy. In water the peptide does not form any regular structure; however, addition of the helix-inducing solvent trifluoroethanol (TFE) causes it to form an alpha-helical structure. The proton NMR spectra of this peptide in 25% and 40% TFE were assigned by double quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect correlated spectroscopy spectra. In addition, the alpha-carbon chemical shifts were obtained from (1H,13C)-heteronuclear multiple quantum coherence spectra. The presence of numerous dNN(i, i + 1), d alpha N(i, i + 3), and d alpha beta(i, i + 3) NOE crosspeaks indicates that an alpha-helix can be formed from residues 3 to 20; this is further supported by the CD data. Upfield alpha-proton and downfield alpha-carbon shifts in this region of the peptide provide further support for the formation of an alpha-helix. The helix induced by TFE appears to be similar to that formed upon binding of the peptide to CaM.

Amino Acid Sequence↗
PubMed1993

[The isolation and identification of a Clostridium botulinum serotype A strain].

319 soil specimens were collected from different places of China for isolating Clostridium botulinum. A strain of Clostridium botulinum was isolated from a culture of soil specimens in Ruoergai of Sichuan Province, the strain was called As-3. The As-3 was identified as Clostridium botulinum serotype A according to its biological properties, biochemical serological and toxicological characteristics and DNA determination. Its DNA G + C mol is 24.9%. The toxin produced by As-3 strain can only be neutralized by type A antiserum.

Animals↗
PubMed1992

Mass culture of anchorage-dependent animal cells with newly born calf skin collagen membrane and microcarrier.

It was shown that collagen substrate enhances the growth as well as the differentiation of many cells in culture. Collagen is one of the major fibrous proteins of animal bodies and the pure collagen used in this experiment was extracted from the newly born calf skin by chemical and biochemical methods. The analytical results obtained by ion-exchange chromatography and electrophoresis showed that the main components of denatured collagen were alpha monomers and beta dimers. The collagen and denatured collagen membranes were prepared by coating their solution on peteri dishes. Various types of cells were cultured on these membranes after being irradiated by ultraviolet ray. The denatured collagen was proved a good substratum for culturing anchorage dependent cells. A denatured collagen (gelatin) microcarrier, GT-2 was obtained by cross-linking gelatin with glutaraldehyde in a suspension polymerization process. These microcarriers were used successfully to culture various anchorage-dependent cells such as Vero, CHO, Bowes and fish cells in varying scales, including T-flasks, spinning bottles, revolving bottles and 1.5 l and 20 l bioreactors.

Animals↗