Abstract
Subtilisin BPN’ (BPN) is an industrially important serine protease that has been extensively investigated in many laboratories. In an effort to improve the thermal stability of the enzyme, researchers at Procter & Gamble have used site-directed mutagenesis techniques to produce several variants of BPN in which residues at the surface of the enzyme have been substituted. We initiated crystallographic studies to determine the structural consequences of these amino acid substitutions. In the course of this work we obtained excellent crystals that correspond to the C2 crystal form of native BPN that has been previously reported.1 Since the structure reported in that work was of only medium resolution, high-resolution X-ray data for this crystal form of native BPN have been collected and the refinement of the structure has been extended using these new data. Isomorphous crystals of two variants, Q19E and Q271E, have also been grown, high-resolution X-ray data have been collected for these crystals, and the experimental results are described. The structures of the native enzyme and the Q271E variant have been refined and are described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
C.S. Wright, R.A. Alden, and J. Kraut, Nature(London) 22: 235 (1969).
P.M. Colman, J.N. Jansonius, and Matthews, J. Mol. Biol.70: 701 (1972).
M.F Perutz and H. Raidt, Nature(London) 255: 256 (1975).
M.F Perutz, Science201: 1187 (1978).
C.R. Erwin, B.L. Barnett, J.D. Oliver, and J.R Sullivan, Prot. Eng.4: 87 (1990).
E.A. Stura and I. A. Wilson, in:“Crystallization of Nucleic Acids and Proteins: A Practical Approach”, A. Ducraix and R. Giege, eds., IRL Press, Oxford, p. 107 (1992).
P.M.D. Fitzgerald, J. Appl. Crystallogr.21: 273 (1988).
R. Bott, M. Ultsch, A. Kossiakoff, T. Graycar, B. Katz, and S. Power, J. Biol. Chem.263: 7895 (1988).
B.C. Finzel, J. Appl. Crystallogr.20: 53 (1987).
W.A. Hendrickson and J.H. Konnert, in:“Biomolecular Structure, Conformation, Function, and Evolution”, R. Srinivasan, ed., Pergamon Press, Oxford, Vol. 1, p. 43 (1980).
W. Furey, B.C. Wang, and M. Sax, J. Appl. Crystallogr.15: 160 (1982).
G. Gilliland, D.T. Gallagher, P. Alexander, and P. Bryan, this volume.
R. Bott, J. Dauberman, L. Wilson, G. Ganshaw, H. Sagar, T. Graycar, and D. Estell, this volume.
A.P. Heiner, H.J.C. Berendsen, and W.F. van Gunsteren, Proteins: Struct. Funct. Genet.14: 451 (1992).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Plenum Press, New York
About this chapter
Cite this chapter
Oliver, J.D., Rydel, T.J., Strickland, L.C. (1996). Crystallographic Investigations of Subtilisin BPN’ Mutants Engineered for Studying Thermal Stability. In: Bott, R., Betzel, C. (eds) Subtilisin Enzymes. Advances in Experimental Medicine and Biology, vol 379. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-0319-0_21
Download citation
DOI: https://doi.org/10.1007/978-1-4613-0319-0_21
Publisher Name: Springer, Boston, MA
Print ISBN: 978-0-306-45108-9
Online ISBN: 978-1-4613-0319-0
eBook Packages: Springer Book Archive