[VCP] Vaccinia Complement Protein

[VCP] Vaccinia Complement Protein

The intention of this module is introduce you to the applications of structural biology by utilizing a practical example, namely, Vaccinia Complement Protein (VCP), and the opportunity to use modern, robotic protein purification equipment.

Syllabus

  1. Structure of VCP
    1. Mapping and primary structural similarity.
    2. Comparison of VCP to the family of complement control protein.
    3. Demonstration of Over-Expression of VCP.
    4. NMR structure of VCP.
    5. X-ray structure of VCP.
  2. Structure-function relationship of VCP
    1. Biological functions of VCP.
    2. Discussion and demonstration of truncation and site specific mutations of VCP.
    3. Assignment of essential domains for binding to complement components, heparin and suramin.
  3. Structure-stability relationship of VCP
    1. Influence of extremes of pH, temperature, freeze thawing, storage and lyophilization on VCP.
    2. Structural changes and melting curves with adverse physical conditions.

Online Lectures/Resources

References

Kotwal, GJ and B Moss. 1988. "Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant." Virology, 167 (2): 524-537.

Kotwal, GJ and B Moss. 1988. "Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins." Nature, 335 (6186): 176-178.

Kotwal, GJ, SN Isaacs, R McKenzie, MM Frank, and B Moss. 1990. "Inhibition of the complement cascade by the major secretory protein of vaccinia virus." Science, 250 (4982): 827-830.

Kotwal, GJ. 1994. "Purification of virokines using ultrafiltration." American Biotechnology Laboratory, 12 (10): 76-77.

Kotwal, GJ. 2000. "Poxviral mimicry of complement and chemokine system components: what's the end game?" Trends in Immunology, 21 (5): 242-248.

McKenzie, R, GJ Kotwal, B Moss, CH Hammer, and MM Frank. 1992. "Regulation of complement activity by vaccinia virus complement-control protein." Journal of Infectious Diseases, 166 (6): 1245-1250.

Murthy, KH, SA Smith, VK Ganesh, KW Judge, N Mullin, PN Barlow, CM Ogata, and GJ Kotwal. 2001. "Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans." Cell, 104 (2): 301-311.

Smith, SA, NP Mullin, J Parkinson, SN Shchelkunov, AV Totmenin, VN Loparev, R Srisatjaluk, DN Reynolds, KL Keeling, DE Justus, PN Barlow, and GJ Kotwal. 2000. "Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells." Journal of Virology, 74 (12): 5659-5666.

Smith, SA and GJ Kotwal. 2001. "Virokines: novel immunomodulatory agents." Expert Opinion on Biological Therapy, 1 (3): 343-357.

Smith, SA, G Krishnasamy, KHM Murthy, A Cooper, K Bromek, PN Barlow, and GJ Kotwal. 2002. "Vaccinia virus complement control protein is monomeric, and retains structural and functional integrity after exposure to adverse conditions." Biochimica et Biophysica Acta, 1598 (1-2): 55-64.

Smith, SA, R Sreenivasan, G Krishnasamy, KW Judge, KH Murthy, JS Arjunwadkar, DR Pugh, and GJ Kotwal. 2003. "Mapping of regions within the vaccinia virus complement control protein involved in dose-dependent binding to key complement components and heparin using surface plasmon resonance." Biochimica et Biophysica Acta, 1650 (1-2): 30-39.

Wiles, AP, G Shaw, J Bright, A Perczel, ID Campbell, and PN Barlow. 1997. "NMR studies of a viral protein that mimics the regulators of complement activation." Journal of Molecular Biology, 272 (2): 253-265.