Effect of residue 65 substitutions on thermal stability of tissue plasminogen activator kringle-2 domain.
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 (melting temperature), 81 +/- 5 kcal/mol for delta H g, and 1.2 +/- 0.9 kcal/(mol-deg) for delta C p. Thermal denaturation of w-t kringle-2 is reversible in the pH range 3-6 as indicated by the observation of similar heat capacity profiles for consecutive heating cycles and also recovery of spectroscopic and lysine binding properties upon cooling the heat-denatured protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Kelley
R F
RF
Biomolecular Chemistry Department, Genetech, Inc., South San Francisco, California 94080.
Cleary
S
S
D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004795:Enzyme Stability; D014644:Genetic Variation; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014633:Valine
D010959:Tissue Plasminogen Activator; D014633:Valine