[Regulation of skeletal ryanodine receptor by calcium].
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Calcium↗
Biomedical subjects
Chang-Cheng Yin
Publications and source records attributed to Chang-Cheng Yin.
Physical coupling between ryanodine receptor-calcium release channels.
Ryanodine receptor-calcium release channels play a pivotal role in the calcium signaling that mediates muscle excitation-contraction coupling. Their membrane organization into regular patterns, functional gating studies and theoretical analysis of receptor clustering have led to models that invoke allosteric interaction between individual channel oligomers as a critical mechanism for control of calcium release. Here we show that in reconstituted "checkerboard-like" lattices that mimic in situ membrane channel arrays, each oligomer is interlocked physically with four adjacent oligomers via a specific domain-domain interaction. Direct physical coupling between ryanodine receptors provides structural evidence for an inter-oligomer allosteric mechanism in channel regulation. Therefore, in addition to established cytosolic and luminal regulation of function, these observations indicate that channel-channel communication through physical coupling provides a novel mode of regulation of intracellular calcium release channels.
Animals↗
Two-dimensional crystallization of the ryanodine receptor Ca2+ release channel on lipid membranes.
The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 x 10(3) kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a 'trial-and-error' strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ < or = 4 extending to approximately 20 angstroms, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role.
Amino Acid Sequence↗
[Progress in the study of ryanodine receptor-related muscle diseases].
Ryanodine receptor (RyR), a calcium release channel within the sarcoplasmic reticulum (SR) membrane, is a key protein in skeletal and cardiac excitation-contraction coupling. The structural and functional changes of RyR can result in decoupling of excitation-contraction, and lead to muscle diseases. Current study confirms that RyR is related to the pathogenesis of these diseases. In this review, we look at the current progress in the study on RyR and these diseases to provide the molecular basis in the prevention and therapy of these diseases.
Animals↗
[A preliminary study of the structure prediction and expression of SARS-CoV spike protein].
In this study, the encoding sequences of SARS-CoV spike protein were analyzed by bioinformatics methods, the structural characteristics and functions were forecasted based on available data. It suggests that the fragment of spike glycoprotein (S401-659) may be crucial for viral attachment and may be a major immunodominant epitope. Then the fragment was amplified and subcloned into expression vector pET28a(+) and pPIC9K. These two plasmids pET28a(+)-S and pPIC9K-S were transformed to E.coli strain BL21(DE3)-star and Pichia pastoris, respectively. SDS-PAGE and Western blot analysis showed that the recombinant protein was successfully expressed. The denatured inclusion bodies were purified with Ni-NTA chelate agarose and its purity can reach 90%.
Blotting, Western↗
A new approach for rapidly reshaping single-chain antibody in vitro by combining DNA shuffling with ribosome display.
Antibody reshaping is an effective way to reduce the immunogenicity while maintaining or improving the affinity of murine antibodies. This paper describe a new in vitro approach for rapidly reshaping murine antibodies by combining DNA shuffling with ribosome display. With the new method, a reshaping anti-4-1BB single-chain antibody (scFv), Re-4B4-1 scFv, which bound to its antigen (4-1BB) specifically and strongly, was selected from a reshaping library. These results proved definitely the feasibility of the new designed approach for antibody reshaping.
Amino Acid Sequence↗
Soluble expression of recombinant human secondary lymphoid chemokine (SLC) in E. coli and research on its in vitro and in vivo bioactivity.
Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that plays an important role in leukocytes homing to lymphoid tissues. The ability of SLC to co-localize both T cells and dendritic cells formed the rationale to evaluate its utility in cancer immunotherapy. The in vivo antitumor effect of murine SLC (mSLC) has been well documented, but little is known about that of human SLC (hSLC). To investigate the antitumor efficiency in vivo of hSLC, the hSLC gene was artificially synthesized and induced to express as a soluble form in Escherichia coli. After purification, the purity of the recombinant human SLC (rhSLC) protein was above 95% by SDS-PAGE analysis. The K(d) of rhSLC binding to peripheral blood lymphocytes (PBLs) was 0.2186 +/- 0.02675 microM as assessed by FACS, and the maximal chemotactic index of rhSLC was 9.49 at 100 nM as assessed by in vitro chemotaxis assay. Then genomic sequences of hSLC and mSLC, and of human CCR7 (hCCR7) and murine CCR7 (mCCR7), the receptor for SLC, were aligned. It was found that hSLC and mSLC share 70.72% identity and hCCR7 and mCCR7share 86.77% identity. Furthermore, we found that rhSLC could chemoattract murine peripheral blood mononuclear cells (PBMCs) in vitro. On the basis of these facts, immune competent mice inoculated with S180 sarcoma cells were chosen as an in vivo model. Intratumoral injections of rhSLC inhibited tumor growth and increased survival. These findings suggest that, despite its incapability to bind to either human or murine CXCR3, which is related to angiostasis, rhSLC can induce an antitumor response in vivo by another route. This report proves that rhSLC has a potent tumor-inhibition ability that makes it a promising candidate agent in cancer immunotherapy.
Adult↗
[An efficient approach to the synthesis of a high-quality random peptide repertoire].
During the construction of a random peptide repertoire using degenerate models, unexpected amino acids or stop codons are almost unavoidable. To conquer this shortcoming, a new split-mix-split method of oligonucleotide synthesis was developed. A 13-amino acids random peptide library had been constructed by using this method. The sequencing results of 16 clones indicated that neither stop codon nor codon for cysteine appeared as designed. The occurrence rations of 19 amino acids were also calculated and no obvious amino acid bias had been observed. By using this method, the type and quantity of amino acid at certain position of a peptide could be controlled well, so this split-mix-split method, combined with degenerate could meet the needs of a high diversity random peptide library.
Amino Acid Sequence↗
Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli.
Single-chain Fv antibodies (scFv), a group of reconstructed molecules with several disulfide bonds, are prone to aggregate as inclusion bodies, the insoluble species of natural proteins, when expressed in Escherichia coli, especially at high level. Recovery of functionally active products from inclusion bodies is onerous and ineffective. We have increased the soluble and functional scFv yields by fusing either DsbC or DsbG, two E. coli disulfide isomerases with general chaperone function, to scFvs. Compared to the totally insoluble inclusion bodies of scFvs expressed separately, more than half of each fusion protein DsbC-scFv or DsbG-scFv was soluble, according to SDS-PAGE analysis. The more effective solubility was obtained when the fused protein DsbG-scFv was co-expressed simultaneously with DsbC under the same promoter. Under this condition, the soluble portion of DsbG-scFv increased from about 50% to 90% measured by scanning SDS-PAGE gel. Co-expression of DsbC can change fusion protein CBD-scFv from totally insoluble when expressed in E. coli separately to a considerable portion of soluble CBD-scFv. Antigen-binding activity assay showed that scFvs retained full affinity to specific antigens. We also determined that general molecular chaperones GroEL and GroES had no effects on the solubility of scFvs when co-expressed with scFv in E. coli. We propose that the correct formation of disulfide bonds in scFvs is the crucial factor responsible for solubility of scFvs.
Base Sequence↗