Circular beta-lactamase: stability enhancement by cyclizing the backbone

We have cyclized the polypeptide backbone of beta-lactamase with a short pe ptide loop as a novel method for protein stabilization, using intein-mediat ed protein ligation, Successful cyclization was proven by mass spectrometry and subsequent relinearization by proteolytic cleavage, as well as by resi stance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of beta-lactamase was found t o be significantly more stable against irreversible aggregation upon heatin g 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 exopeptidas e-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 disul fide bonds for protein stabilization follows the same rationale, the cycliz ation strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proxi mity.