Structural characterization of the urea-unfolded state of Colicin Immunity Protein Im7 and Im9

Dissertação para obtenção do Grau de Mestre em Bioquímica Estrutural e Funcional

Most single domain proteins have the ability to fold spontaneously into a precise, functional three-dimensional structure in seconds or less. Understanding how this transition occurs will not only help to uncover the way in which an amino acid sequence encodes the corresponding structure but is also likely to provide insight into the folding/unfolding transitions that many proteins undergo as part of their normal functioning. The characterization of these states is particularly important because they often play crucial roles in folding and misfolding processes, responsible for many human neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. In this work, two well characterized proteins, Colicin immunity proteins Im7 and Im9, where used as model for a structural study involving two different approaches to promote their denaturation/unfolding. Im7 and Im9 share a high sequence and structural homology, but despite that fact they fold with different kinetic mechanism in vitro. By using 1H-15N HSQC spectra as a main tool, we have undertaken a comparative study to identify the residues more affected during the denaturation process of Im7 and Im9 promoted by a physical effect, temperature increase, and by a chemical agent, urea. Our aim was to detect possible similarities that could give insight into the aspects that govern folding/unfolding transitions. The results from the temperature study show that the residues most perturbed with increasing temperature are mostly located in loop regions between helices, while urea targets preferably residues that are accessible and solvent exposed. Our study, points out that the ends of well-structured helices can concertedly unfold without entering the mid region residues in the same unfolding process. There seems to be a correlation between dynamic residues (most affected by temperature) and the residues in the regions most perturbed by urea. The results shown that entire loop regions on both proteins may act as concerted units during the unfolding process, and contribute for favorable interactions that delimit and stabilize native-like structural features on the urea-unfolded state, allowing buried regions to be less solvent exposed.

Fundação para a Ciência e a Tecnologia - PEst-C/EQB/LA0006/2011 and Project no. PTDC/QUI-QUI/098892/2008