Reconstructing the lactamase fold with secondary-structure, spare sequences.

RISSO VA; PRIMO ME; SICA MP; GEBHARD LG; ERMÁCORA MR

Montevideo (Uruguay)

Congreso; 34th Annual Meeting of the Argentinean Biophysical Society; 2007

Assembly of portions of protein structure     Our previous works on protein folding determinants, has demonstrated that after permutation and/or terminal deletion b‑lactamase of Bacillus licheniformis (ESBL),no particular segment arises as essential for the codification of the global ESBL structure. These results are congruent with the notion that local structural information and its modular organization can impart most of the tertiary fold specificity and cooperativity. In this work the existence of information modules embedded in the polypeptide chain was studied, replacing portions of the ESBL with paradigm sequences. These sequences belong to proteins non‑related to the ESBL and are very prone to form elements with secondary structure. The proteins variants were produced in E. coli cells and purified. The structure content of the variants was analyzed by circular dichroism (CD). The hydrodynamic properties and the aggregation state were studied by molecular exclusion chromatography (SEC-FPLC), and their cooperativity was studied by thermal denaturation followed by CD signal at 220 nm. Most frequently, the variants were observed in a soluble dimeric state. In accordance to other results, all variants were highly susceptible to the proteolysis, indicating absence of rigid structure. Notwithstanding, observed catalytic constants revealed existences of the native fold.