PMID: 2502177May 2, 1989Paper

Effect of residue 65 substitutions on thermal stability of tissue plasminogen activator kringle-2 domain

Biochemistry
R F Kelley, S Cleary

Abstract

We have used differential scanning calorimetry to measure the effect of replacements of valine 65 on thermal stability of the isolated kringle-2 domain of tissue plasminogen activator (t-PA). The role of this site in stability was examined because a human t-PA variant having this valine (residue 245 in t-PA numbering) replaced with a methionine has been described [Johnston, M.D., & Berger, H. (1987) U.K. Patent Application GB 2176702A]. Mutants of kringle-2 having valine 65 replaced with Met, Leu, Ile, Thr, Ala, or Ser were constructed by using site-directed mutagenesis in conjunction with a restricted site selection strategy. Isolated kringle-2 domains were expressed in Escherichia coli and purified as previously described for the wild-type domain [Cleary, S., Mulkerrin, M.G., & Kelley, R.F. (1989) Biochemistry 28, 1884-1891]. None of these substitutions results in a significant perturbation of the native conformation of kringle-2 as judged by far-UV circular dichroism and equilibrium dialysis measurements of L-lysine affinity. A two-state analysis of the heat capacity profile observed for heating a solution of wild-type (w-t) kringle-2 containing 100 mM citrate, pH 4.5, provides values of 64.3 +/- 0.8 degrees C for Tg (meltin...Continue Reading

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Citations

Jul 17, 1999·The Journal of Peptide Research : Official Journal of the American Peptide Society·Y ChangF J Castellino
Apr 1, 1992·Archives of Biochemistry and Biophysics·V S DeSerranoF J Castellino
Apr 1, 1992·The American Journal of Medicine·C S KaseJ K LittleJohn
May 19, 2000·Methods : a Companion to Methods in Enzymology·V A Ploplis, F J Castellino

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