Resumen de Obtención de moléculas lineales de AS-48 que conserven la estructura tridimensional y la actividad biológica de la molécula nativa
AS-48 is a 70-residue circular peptide from Enterococcus faecalis belonging to a heterogeneous group of bacterial antimicrobial polypeptides, called bacteriocins. The complete NMR assignment indicated that the AS-48 fold is characterised by five helices arranged to form a globular structure with a hydrophobic surface located in helices a1-a3 and a highly positive charged region concentrated in helices a4 and ¿5.
The circular backbone of AS-48 has prompted us to explore the possibility of its linearization. In a first attempt, we decided to open it between Met1 and Trp70 and between the two wider loops situated between the a1/a2 and a3/a4 helices by applying genetic circular permutation. In this way, the as-48A structural gene derivatives obtained (as-48L1/70, as-48L23/24 and as-48L48/49) were cloned into different vector to be expressed in E. coli strains, including those with protease-deficient activities. Unfortunately, no linear proteins with the expected size could be observed in the cell extracts, under any of the induction conditions assayed, in spite of transcription from the recombinant genes was detected. A plausible justification for this behaviour could be that the disruption of the circular AS-48 backbone at such sites might be involved in topological and entropic changes that disturb the potential conformation of the linear derivatives, rendering them incapable of folding into native-like structures or at least into stable structures and being consequently degraded by the host proteases. To amplify the E. coli gene expression capacity, as-48L23/24 and as-48L48/49 linear permutants were separately cloned in frame with the C-Lytag protein, which is controlled by linked regulatory circuits in the pALEXa vector. This approach provided fused proteins with the expected size (approx. 29000 Da) in each induced cell extract, which were recognized by specific antisera. Interestingly, unspecific enterokinase cleavage release two hybrid proteins, corresponding to fusions of tag fragments with partial sequences of AS23/24 and AS48/49 derivatives respectively that exhibited antibacterial activities.
In addition, we have conducted limited proteolysis experiments on AS-48 to produce linear forms, considering that this technique allows the production of linear derivatives able to maintain a native-like conformation. In fact, we report here the production, after thermolysin treatments under denaturing conditions, of a protein species carrying a single nicking (AS10/11) and fragments of 55- and 38-residues (AS43-27 and AS42,43-10) that retain partly a-helical secondary structure and the biological activity of the intact protein. The most important conclusion of these results has been to confirm that circularization is not essential for the bactericidal activity but to stabilise the three dimensional structure of the protein. It has been also demonstrated that is important to maintain a certain distribution of electrostatic and hydrophobic surfaces for an efficient AS-48 insertion into the membranes. This observation suggests that nicking is compatible with the insertion into the membrane that is required for the activity.
