title: Circular beta-lactamase: stability enhancement by cyclizing the backbone.
abstract: We have cyclized the polypeptide backbone of beta-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 beta-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.
authors.affiliation: Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland.
authors.forename: H
authors.initials: H
authors.lastname: Iwai
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010456:Peptides, Cyclic; D010447:Peptide Hydrolases; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1