Systematic deletions of sequence to study protein folding

GEBHARD LEOPOLDO G.; RISSO VALERIA A.; SANTOS JAVIER; FERREYRA RAUL G.; NOGUERA MARTÍN EZEQUIEL; ERMÁCORA MARIO ROBERTO

Rosario

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

To identify the amino acids residues critical for the 3D structure of an enzyme, we carried out a systematic segmental deletion through the amino acid sequence of Bacillus licheniformis b-lactamase[1,2]. Combining circular permutation and terminal truncation, we constructed 27 abridged variants lacking fragments of 5 21 residues along the sequence up to a formal coverage of 87.5% (231/264) of the residues. Both optical and hydrodynamic analysis revealed that most variants form complex mixtures containing large amounts of misfolded protein. However, enzymatic assays showed that all the variants performed enzymatic activity to a greater or lesser extent. Catalysis by the less active variants was studied further. HPLC analysis of the transient acyl enzyme demonstrated that in these variants the more affected step in catalysis was the deacylation step but substrate recognition and the nucleophilic attack of Ser70 are native like. Our results suggest that no segment of the b-lactamase sequence is essential to reach the native conformation, which is compatible with the idea that the tertiary fold specificity and cooperativity are mostly determined by local information and modular organization. References: [1] Gebhard, L.G., Risso, V. A., Santos, J., Ferreyra, R. G., Noguera, M. E. and Ermácora, M. R., Journal of Molecular Biology, 2006, 358, 280 - 288. [2] Santos, J., Gebhard, L. G., Risso, V. A., Ferreyra, R. G, Rossi, J. P. F. C. and Ermácora, M. R., Biochemistry, 2004, 43, 1715 - 1723.