Deletion reveals sequence segments nonessential for proper folding of B. licheniformis b-lactamase

FERREYRA RAÚL G.; GEBHARD LEOPOLDO G.; SANTOS JAVIER; RISSO VALERIA A.; NOGUERA MARTÍN E.; ERMÁCORA MARIO R.

Mar del Plata, Argentina

Congreso; Congreso conjunto de Sociedades Biomédicas; 2004

Exo small b-lactamase (Knox and Moews, 1991), a 265-residue protein with no cysteine, is organized in two closely associated noncontinuous domains. One of these domains includes 37 N-terminal and 76 C-terminal amino acid residues and is organized in a five-stranded anti parallel b-sheet with one face covered by three amphipathic a-helices. The other domain, comprising the central 152 amino acid residues, folds in a globular array of helical elements that packs against the second face of the b-sheet. The active site is located in the interface between domains. The role of a sequence segment in determining protein structure and activity can be assessed by genetic engineering. We have observed that deletion of the last nine residues from exo small b-lactamase (ES-bL) yields a molecule that folds properly into a native monomeric state with catalytic parameters that are similar to those of wild-type enzyme. Despite the low impact on the structure, this deletion affects dramatically the rate of folding: the truncated variant folds 104 times slower than the wild-type protein (Santos et al., 2004). Using an approach based on circular permutation of the polypeptide chain (Pieper et al., 1997), we prepared bLcp117/95, bLcp179/162, bLcp227/216 and bLcp277/266, which are variants of ES-bL circularly permuted and truncated from the new termini to delete a fragment of 10-21 residues. Since these removed segments are on the protein surface, the deletions produce no internal cavities that may complicate the interpretation of folding experiments, and it leaves specific tertiary contacts removed as the cause of the observed effect. The current study examines the impact of the deletions on the activity and structure of the ES-bL.