Protein Dynamics and Proteolytic Susceptibility

Proteins play a central role in all fundamental processes of life. Their life span corresponds to their function. Proteins, which are responsible for signal transduction exhibit a short life span, whereas structural proteins are characterized by a slow turnover rate. Proteases are enzymes digesting proteins. Thus, the susceptibility to protease cleavage is decisive for the life span of proteins. Interactions between proteins and proteases required for digestion rely on a specific, partially unfolded structure of the protein. Hence, a folded protein needs to exhibit a tendency for substantial unfolding to be digested. This project aims at investigating possible relations between proteolytic susceptibility, unfolding tendency and the inherent structural dynamics of proteins. Computer simulations are an efficient and reliable method do characterize dynamics of proteins. The major birch pollen allergen Bet v 1 is an optimal model system for our investigations. On the one hand 40 % of all allergy patients in the western hemisphere are affected by an allergy to birch pollen. On the other hand, there is a substantial amount of high quality experimental data available in the literature. Furthermore, we work in close collaboration with several experts in the field of pollen allergy, providing us access to unpublished experimental results to develop and validate our models. Even though different isoforms of Bet v 1 have a high identity in sequence and structure, they vary considerably in their allergenic potency. There are strong hints in the literature and in our preliminary results suggesting a correlation between structural dynamics, proteolytic susceptibility and allergenic potency of Bet v 1. We envisage that by profiling dynamics of Bet v 1 our research allows to develop a broad understanding for the molecular determinants of its allergenic potency. The resulting insights are expected to contribute to the development of safer and more efficient allergy treatment strategies.

Proteolytic degradation is vital in countless biological processes, such as signal transduction, nutrition and regulation of immune response. Depending on its function a protein needs to be either resistant or susceptible to protease recognition and cleavage. Numerous descriptors have been proposed to explain the origin of strongly varying rates at which different proteins are digested including sequence, size, charge, N-terminal residue and flexibility. In this project, we focused on the proteolytic processing of the major birch pollen allergen Bet v 1 and possible implications for its allergenicity. Bet v 1 is an ideal model system since its proteolytic stability, allergenic potency, structure and dynamics have been thoroughly investigated over the last decades, resulting in a considerable amount of high-quality data. The molecular determinants of the allergenic potency of a protein are obscure. It is unclear why some proteins cause a strong allergic response in genetically predisposed individuals, whereas proteins of almost identical fold are well tolerated. A key step in allergen processing is fragmentation by endosomal proteases. Proteolytic susceptibility, being a prerequisite for degradation, is thus decisive for the allergenic potency of a protein. In Bet v 1 most protease cleavage sites are located within secondary structure elements. Yet, the involved proteases exclusively recognize substrates in extended beta-sheet conformations. Hence, the folded protein has to undergo major conformational rearrangements to a partially unfolded state to become susceptible to proteolysis. Our scope was to characterize molecular determinants, which regulate the proteolytic susceptibility, focusing on conformational flexibility. We have performed molecular dynamics (MD) simulations using classical as well as enhanced sampling techniques to cover protein dynamics from the micro- to millisecond time scale. Additionally, effects of variations of temperature and pH on the captured conformational ensemble have been investigated.