julian.fuchs

Stipendium: 
DOC
Fachbereich: 
Natur- und Biowissenschaften, Medizin
Porträtbild: 
Projekttitel: 
Local Dynamics in Protease Recognition
Studienrichtung: 
Theoretische Chemie
Forschungsstätte: 
Leopold Franzens Universität Innsbruck
Förderdauer: 
01.01.2012 - 31.12.2014
Schlagworte: 
Protease, Flexibilität, Spezifität, Dynamik, Serinprotease, Molecular Dynamics, NMR

Proteases catalyze cleavage of peptide bonds and are involved in virtually all fundamental cellular processes. Far over 500 proteases with unique substrate cleavage patterns have been identified in the human genome. These patterns reach from specificity for a single peptide to broad spectra of cleaved peptides. For instance, digestive enzymes are known to process a wide range of substrate sequences in contrast to proteases involved in signaling pathways cleaving only very distinct peptide bonds.
Substrate recognition rules for proteases focus on distinct molecular interactions mainly in the S1 binding pocket between protease and substrate. However, established rules generally fail to consider the inherent flexibility of biomolecules. It is generally accepted, that enzymes in aqueous solution undergo conformational transitions, which are not taken into account in the historic “lock-key” binding model of Fischer. In contrast, a modern binding mechanism model like “conformational selection” considers conformational ensembles of the receptor. It proposes a mechanism with a substrate selecting its appropriate binding conformation from pre-existing conformations of the receptor.
Experimental and computational techniques will elucidate local dynamics of proteases in the binding site region. Following the model of conformational selection, we expect to observe correlations between intrinsic flexibility of proteins and substrate specificity. We surmise that proteases providing a larger conformational ensemble cleave a broader substrate spectrum. A close interplay between molecular dynamics simulations and NMR experiments will allow us to assess differences in local dynamics in structurally similar and homologous serine proteases. In parallel, analyses of existing protease cleavage databases will provide activity data and permit to elucidate the impact of local binding site dynamics on protease specificity.
Multiple molecular dynamics simulations with starting points from publicly available high resolution X-ray structures will be performed to investigate serine protease flexibility at an atomistic level. Therefore, flexibility of the substrate binding regions will be quantified and correlated to available substrate sequence-dependent cleavage data. Additionally, NMR experiments will support in silico findings by the experimental determination of order parameters giving insights into residue-wise contributions for protein dynamics.
Ultimate goal of the project is to develop a model linking local protease dynamics to respective cleavage specificity complementing established rules for serine protease specificity based on P1-S1 interactions. These findings will provide a deeper understanding of cellular regulation processes involving proteases as well as starting points for structure-based development of new pharmaceutical compounds targeting distinct proteases.