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Circular β‐lactamase: stability enhancement by cyclizing the backbone
Author(s) -
Iwai Hideo,
Plückthun Andreas
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01220-x
Subject(s) - chemistry , cleavage (geology) , disulfide bond , stereochemistry , peptide , protein structure , carboxypeptidase , crystallography , biophysics , biochemistry , enzyme , biology , paleontology , fracture (geology)
We have cyclized the polypeptide backbone of β‐lactamase with a short peptide loop as a novel method for protein stabilization, using intein‐mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent re‐linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of β‐lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his‐tag which became exopeptidase‐resistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N‐ and C‐termini in close proximity.