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Peptide rescue of an N‐terminal truncation of the stoffel fragment of Taq DNA polymerase
Author(s) -
Vainshtein Inna,
Malcolm Bruce A.,
Atrazhev Alexey,
Elliott John F.,
Eom Soo Hyun,
Wishart David S.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560050904
Subject(s) - polymerase , dna polymerase , thermus aquaticus , amino acid , taq polymerase , microbiology and biotechnology , peptide , peptide sequence , biology , thermostability , biochemistry , chemistry , dna , enzyme , gene
Deletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5′ to 3′ exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N‐terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the StofΔ12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the StofΔ47, in which 47 amino acids were deleted), resulted in a significant loss of both activity and thermostability. A 33‐amino acid synthetic peptide, based on this critical region (i.e., residues 303–335 inclusive), was able to restore 85% of the StofΔ12 activity when added back to the truncated StofΔ47 protein as well as return the temperature optimum to that of the StofΔ12 and Stoffel proteins. Examination of the crystal structure of Taq polymerase (Kim et al., 1995) shows that residues 302–336 of the enzyme form a three‐stranded β‐sheet structure that interacts with the remainder of the protein. CD analysis of the 33‐amino acid peptide indicates that the free peptide also adopts an ordered structure in solution with more than 50% β‐sheet content. These data suggest that this 33‐amino acid peptide constitutes a stable β‐sheet structure capable of rescuing the truncated polymerase in a fashion analogous to the well‐documented complementation of Ribonuclease S protein by the 15‐residue, α‐helical, S peptide.