Research

[Recombinant human CXCL8(3¡V72)K11R/G31P] [Antimicrobial peptide]
[Dengue virus envelope protein][Japanese encephalitis virus envelope protein]

[Bruton's Tyrosine Kinase] [Hepatitis delta virus] [DNA/RNA Hybrid]
[Adapter protein-Grb2] [Tetratricopeptide repeat-TPR]


  • Recombinant human CXCL8(3¡V72)K11R/G31P
  • The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases.Among them, CXCL8 (interleukin-8, IL-8), binds to both the CXCR1 and CXCR2 receptors with high affinity and the expression levels of CXCL8 are elevated in many inflammatory diseases. Recently, an analogue of human CXCL8, CXCL8(3¡V72)K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both CXCR1 and CXCR2. To obtain large quantities of hG31P, we have successfully constructed and expressed hG31P in Escherichia coli. Moreover, we have developed a new protocol for high-yield purification of hG31P and for the removal of lipopolysaccharide (LPS, endotoxin) associated with hG31P due to the expression in E. coli. The purity of hG31P is more than 95% and the final yield is 9.7 mg hG31P per gram of cell paste. The purified hG31P was tested by various biological assays. In addition, the structural properties of hG31P were studied by circular dichroism (CD), ultracentrifuge, isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy.

    Related publications:


    1. Hsi-Tsung Cheng, Kuo-Chun Huang, Hui-Yuan Yu, Kun-Jhih Gao, Xixing Zhao, Fang Li, Jennifer Town, John R. Gordon, Cheng JW. "A new protocol for high-yield purification of recombinant human CXCL8(3¡V72)K11R/G31P expressed in Escherichia coli." (2008) Protein Expr Purif. 61(1):65-72.

     

     

     

     

  • Antimicrobial peptide
  • A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH2, designated as Pac-525, was found to possess improved activity against both gram-positive and negative bacteria. We have synthesized two Pac-525 analogues, D-Pac-525 containing all D-amino acids and D-Nal-Pac-525, the D-Pac-525 analogue with tryptophan replaced by D-beta-naphthylalanine. We have determined the solution structure of D-Nal-Pac-525 bound to membrane-mimetic DPC micelles by two-dimensional NMR methods. The DPC micelle-bound structure of D-Nal-Pac-525 adopts a left-hand alpha-helical segment and the positively charged residues are clustered together to form a hydrophilic patch. The surface electrostatic potential map indicates the three D-beta-naphthylalanines are packed against the peptide backbone and form an amphipathic structure. A variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that D-Nal-Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the strong antimicrobial activity of D-Nal-Pac-525 may be due to interactions with bacterial and fungus membranes.

    Related publications:

    1. Yu HY, Huang KC, Yip BS, Tu CH, Chen HL, Cheng HT , Cheng JW. Rational design of tryptophan-rich antimicrobial peptides with enhanced antimicrobial activities and specificities. Chembiochem. 2010 Nov 2;11(16):2273-82.

    2. Wei SY, Wu JM, Kuo YY, Chen HL, Yip BS, Tzeng SR, Cheng JW."Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity." (2006) J Bacteriol. 188 ,328-34.

    3. Wu JM, Wei SY, Chen HL, Weng KY, Cheng HT, Cheng JW. "Solution structure of a novel D-naphthylalanine substituted peptide with potential antibacterial and antifungal activities." (2007) Biopolymers. 88(5):738-45.

     

     

     

     

  • Dengue virus envelope protein
  • Flaviviruses are small (50 nm) positive-strand RNA viruses that contain a lipid-bilayer membrane. Forty species of the flavivirus family have been associated with human diseases and most of them are transmitted to their vertebrate hosts by infected mosquitoes or ticks. Among these, yellow fever (YF), Japanese encephalitis (JE), tick-borne encephalitis (TBE), and dengue (DEN) are the most important viral arboviruses in the world. DEN viral infections affect more than 100 million populations per year. There has been a marked increase of this disease over the last decades associated with significant mortality. Like other flaviviruses, the virion of DEN contains three structural proteins?-?a 12 kD nucleocapsid or core protein (C), a 8 kD nonglycosylated membrane protein (M), and a 53 kD glycosylated envelope protein (E), as well as seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The E protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in viral attachment, fusion, penetration, hemagglutination, host range and cell tropism, and virus virulence and attenuation. The three-dimensional structures of the DENV-2, DENV-3, and TBEV E proteins have been determined by X-ray crystallography.The X-ray structures reveal that the E protein is composed of three discernible domains, Domain I, II, and III. The solution structures of the Domain III of JEV, LGTV, WNV, and DENV-4 envelope proteins have been studied recently. Monoclonal antibodies that bind to Domain III of DENV are found to be the most efficient blockers of virus adsorption to vero cells. However, no detailed spatial configuration of the epitopes of Domain III of DENV E protein has been provided.

    Related publications:

    1. Wu CW, Lin YT, Huang KC, Cheng JW. "1H, 15N and 13C resonance assignments of the domain III of the Dengue virus envelope protein." (2005) J Biomol NMR. 33,76.
    2. Huang KC, Lee MC, Wu CW, Huang KJ, Lei HY, Cheng JW. "Solution structure and neutralizing antibody binding studies of domain III of the dengue-2 virus envelope protein" (2007) Proteins: Structure, Function, and Bioinformatics. Volume 70, Issue 3 ,1116 - 1119.

     

     

     

  • Japanese encephalitis virus envelope protein
  • The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. We have determined the solution structure of the major antigenic domain (domain III) of the Japanese encephalitis virus (JEV) envelope protein. The JEV domain III forms a beta-barrel type structure composed of six antiparallel beta-strands resembling the immunoglobulin constant domain. We have also identified epitopes of the JEV domain III to its neutralizing antibody by chemical shift perturbation measurements. Site-directed mutagenesis experiments are performed to confirm the NMR results. Our study provides a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses.

    Related publications:

    1. K.P. Wu, C.W. Wu, Y.P. Tsao, T.W. Kuo, Y.C. Lou,C.W. Lin, S.C. Wu and J.W. Cheng, " Structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain III of the Japanese encephalitis virus envelope protein. " (2003) Journal of Biological Chemistry 278, 46007-46013.

     

     

     

  • Bruton's Tyrosine Kinase
  • The cytoplasmic tyrosine kinase (Bruton's tyrosine kinase, BTK) was found to play a central role for B cell development. Mutations or deletions within this protein are responsible for X-linked agammagobulinemia (XLA), an inherited immunodeficiency decease. BTK contains an apparent pleckstrin homology (PH) domain, a Src homology 2 (SH2) domain, a SH3 domain and a catalytic tyrosine kinase domain. SH2 and SH3 domains are small protein modules that mediate protein-protein interactoins and are found in many proteins involved in intracellular signal transduction. In order to investigate the role of BTK in B cell development and activation, we are applying multi-dimensional NMR techniques to study structures of the SH2 and SH3 domains of BTK. Peptide libraries will be collected to reveal novel binding ligands for the SH2 and SH3 domains of BTK. Structural studies of the ligand/SH2 and ligand/SH3 complexes will be carried out and new ligands can be designed through a rational approach.

    Related publications:

    1. Stability and Folding of the SH3 Domain of Bruton's Tyrosine Kinase (1996) PROTEINS: Structure, Function, and Genetics 26, 465-471.
    2. SH3 Domain of Bruton's Tyrosine Kinase can Bind to Proline-Rich Peptides of TH Domain of the Kinase and p120cbl (1997) PROTEINS: Structure, Function, and Genetics 29, 545-552.
    3. Solution Structure of the BTK SH3 Domain Complexed with a Proline-Rich Peptide from p120cbl (2000) Journal of Biomolecular NMR 16, 303-312.
    4. Stability and Peptide Binding Specificity of BTK SH2 Domain: Molecular Basis for X-Linked Agammaglobulinemia (2000) Protein Science 9, 2377-2385.
    5. Events of Folding and Dynamics of the BTK SH3 Domain (2001) in preparation.
    6. Solution Structure of the BTK SH2 Domain: Structural Basis for X-Linked Agammaglobulinemia (2000) in preparation.
    7. M.T. Pai, K.C. Huang, S.R. Tzeng, and J.W. Cheng, "1H, 15N and 13C resonance assignments of the SH2 domain of Bruton's tyrosine kinase" (2002) Journal of Biomolecular NMR 24, 163-164.


 

 

 

 

  • Adapter protein-Grb2
  • Growth factors, when binding to the external domain of their receptors, induce oligomerization of receptors, stimulation their protein kinase activity that is responsible for reciprocal transphosphorylation of receptor intracellular domains. The tyrosine phosphorylation sites exhibit a high affinity for SH2 domains (Src Homology 2 domain, ~100 amino acids), the specificity being determined by the residues immediately surrounding the phosphorylated tyrosine. The SH2 domain of Grb2 binds phosphotyrosyl peptides with the consensus sequence pYXNX within several proteins including the adapter proteins SHC, growth factor receptors such as members of the erbB family, morphology-determining proteins such as FAK, and cellular oncogenes such as BCR-abl. Binding of the Grb2 SH2 domain to the receptors relocates the Grb2 SH3 domain binding proteins, i.e. Sos, close to the plasma membrane. Sos, then, due to its guanine nucleotide exchange activity, converts the GDP-bound inactive form of Ras to its GTP-bound active form. Activated Ras triggers the kinase cascade which is essential for cell growth and differentiation. A particularly important role for Grb2 in human cancer has been proposed for cells transformed by high levels of erbB2 (HER-2 or neu) expression. Recent studies have indicated that Grb2 function is required for cell transformation by the neu and bcr-abl oncogenes. Thus, the design of specific inhibitors to Grb2 SH2 domain holds the promise of targeted treatment of breast cancer and cancer. We will use the structure-based drug design (SBDD) strategies to design and synthesize various inhibitors for the Grb2 SH2 domain.

    Related publications:

    1. Y.C. Lou, F.T. Lung, M.T. Pai, S.R. Tzeng, P.P. Roller and J.W. Cheng, "Solution Structure and Dynamics of a Nonphosphorelated Cyclic Peptide Inhibitor for the Grb2 SH2 Domain" (1999) Archives of Biochemistry and Biophysics 372, 309-314.
    2. F.D. Lung, J.Y. Tsai, S.Y. Wei, J.W. Cheng, C. Chen, P. Li, and P.P. Roller, "Novel peptide inhibitors for Grb2 SH2 domain and their detection by surface plasmon resonance" (2002) Journal of Peptide Research 60, 143-149.

     

     

     

     

  • Tetratricopeptide repeat-TPR
  • We will determine the solution structure and dynamics of the Tetratricopeptide repeat motif (TPR) of the Hsc70 associated protein - SGT using NMR spectroscopy. Binding surface and complex structure of the SGT TPR motif and Hsc70 will also be mapped using isotope-edited and filtered NMR experiments.

    Related publications:

    1. M.T. Pai, C.S. Yang, S.R. Tzeng, C. Wang, and J.W. Cheng, "Stability and Folding of the Tetratricopeptide repeat motif of SGT" (2001) submitted.
    2. M.T. Pai, C.S. Yang, S.R. Tzeng, C. Wang and J.W. Cheng, " 1H, 15N and 13C resonance assignments of the tetratricopeptide repeat (TPR) domain of hSGT " (2003) Journal of Biomolecular NMR 26, 381-382.

     

     

     

     

  • Hepatitis Delta Antigen
  • Hepatitis delta virus (HDV) is a satellite of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. The genome of the hepatitis delta virus consists of a single-stranded, circular RNA of 1679 nucleotides which forms a rod structure due to extensive self homology. HDV replicates through synthesis of an antigenomic RNA via a rolling circle mechanism. This mechanism is governed by autocatalytic cleavage and ligation reactions. HDV encodes two proteins, the small delta antigen and the large delta antigen. The latter resembles the former except for the presence in the latter of additional 19 amino acids at the C terminus. While the small delta antigen is required for HDV RNA replication, the large delta antigen inhibits replication. HDV delta antigen differs from other RNA-binding proteins in that this antigen contains multiple regions (residues 2-27, 24-75, 79-107) that mediate RNA binding. In order to study the interaction of hepatitis delta antigen with HDV RNA, we will study the structure of its RNA-binding domain using CD and NMR spectroscopic techniques.

    Related publications:

    1. Y.C. Lou, I.J. Lin, M.T. Pai, and J.W. Cheng, "Solution Structure of an N-Capping Peptide from the N-terminal Leucine-Repeat Region of Hepatitis Delta Antigen" (2000) Archives of Biochemistry and Biophysics 377, 219-227.

    2. I.J. Lin, Y.C. Lou, M.T. Pai, H.N. Wu and J.W. Cheng, "Solution Structure and RNA Binding Activity of the N-terminal Leucine-Repeat Region of Hepatitis Delta Antigen" (1999) PROTEINS: Structure, Function, and Genetics 37, 121-129.

    3. J.W. Cheng, I.J. Lin, Y.C. Lou, M.T. Pai, and H.N. Wu, "Local Helix Content and Nucleic Acid Binding Activity of the N-terminal Leucine-Repeat Region of Hepatitis Delta Antigen" (1998) Journal of Biomolecular NMR 12, 183-188.

     

     

     

  • DNA/RNA Hybrid
  • The solution structure of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)]2, in which the central segment was flanked by DNA duplexes at both ends, was determined using 2D NMR, restrained MD, and NOE back-calculation refinement. Evidence of hydration at different sites in both grooves was found in NOESY and ROESY experiments. Correlation times of hydration and dissociation rate constants between the OH protons and water were measured. The solution structure of this chimeric duplex differed from previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)]2 as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)]2. Overall, the global conformation of this chimeric duplex was closer to its A-RNA analog than to its B-DNA analog . Furthermore, NOEs between water and H1¡Šin the minor groove, which was not observed in its DNA analogue, showed similar identities with its RNA analogue. In the chimeric fragment, no structural parameter of the 5'-end DNA at the DNA¡ERNA hybrid junction was affected by the 3'-end RNA, whereas structural change was found at the 3'-end RNA¡EDNA hybrid junction. This influence was involved in only one step. In contrast to the similarity with its RNA analog, titration of the minor groove binding drug, distamycin A suggested a possible 2:1 binding mode similar with previous DNA-drug binding studies. This result suggested a wider minor width, enough for two parallel-binding mode found in pure DNA duplex. Further structural studies are underway.

    Related publications:

    1. S.T. Hsu, M.T. Chou, and J.W. Cheng, "The Solution Structure of [d(CGC)r(aaa)d(TTTGCG)]2: Hybrid Junctions Flanked by DNA Duplexes" (2000) Nucleic Acids Research 28, 1322-1331.

    2. S.T. Hsu, M.T. Chou, S.H. Chou, W.C. Huang, and J.W. Cheng, "Hydration of [d(CGC)r(aaa)d(TTTGCG)]2" (2000) Journal of Molecular Biology 295, 1129-1137.

    3. M.L. Jain, Y.P. Tsao, N.L. Ho, and J.W. Cheng, "A Facile Synthesis of [N1, NH2-15N2]Adenine, [N3, NH2-15N2]Adenine and [N1, N3, NH2-15N3]Adenine" (2001) Journal of Organic Chemistry in press.

    4. Y.P. Tsao, S.T. Hsu, S.H. Chou, and J.W. Cheng, "The Solution Structure of [d(CGC)r(amamam)d(TTTGCG)]2" (2001) Journal of Biomolecular NMR in press.