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Folding studies
Folding studies
Structural Insights into the Trp‐Cage Folding Intermediate Formation
Structural Insights into the Trp‐Cage Folding Intermediate Formation
Petra Rovó, Pál Stráner, András Láng, István Bartha, Kristóf Huszár, László Nyitray and András Perczel
The 20 residue long Trp‐cage is the smallest protein known, and thus has been the subject of several in vitro and in silico folding studies. In this article, we reported the multistate folding scenario of the miniprotein in atomic detail. We detected and characterized different intermediate states by temperature dependent NMR measurements both at neutral and acidic pH values. We developed a deconvolution technique to characterize the invisible—fully folded, unfolded and intermediate—fast exchanging states. Using nonlinear fitting methods we can obtain both the thermodynamic parameters and the NMR chemical shifts of the conformers of the multistate unfolding process. We observed that during the unfolding of Trp‐cage distinct intermediates evolve: a fast‐exchanging intermediate is present under neutral conditions, whereas a slow‐exchanging intermediate‐pair emerges at acidic pH. Using chemical shift information combined with MD simulations we characterized the structure of the different intermediate states. Our experimental data offer exceptional testing ground for further computational simulations.
This work was done during my PhD at the Eötvös Loránd University in Budapest, Hungary under the supervision of Prof. András Perczel.
Protein Design
Protein Design
Rational design of alpha-helix-stabilized Exendin-4 analogues
Rational design of alpha-helix-stabilized Exendin-4 analogues
Petra Rovó, Viktor Farkas, Pál Stráner, Mária Szabó, Ágnes Jermendy, Orsolya Hegyi, Gábor K. Tóth, András Perczel
Exendin-4 (Ex4) is a potent glucagon-like peptide-1 receptor agonist, a drug regulating the plasma glucose level of patients suffering from type 2 diabetes. The molecule’s poor solubility and its readiness to form aggregates increase the likelihood of unwanted side effects. Therefore, we designed Ex4 analogues with improved structural characteristics and better water solubility. Rational design was started from the parent 20-amino acid, well-folded Trp cage (TC) miniprotein and involved the step-by-step N-terminal elongation of the TC head, resulting in the 39-amino acid Ex4 analogue, E19. Helical propensity coupled to tertiary structure compactness was monitored and quantitatively analyzed by electronic circular dichroism and nuclear magnetic resonance (NMR) spectroscopy for the 14 peptides of different lengths. Both 15N relaxation- and diffusion-ordered NMR measurements were established to investigate the inherent mobility and self-association propensity of Ex4 and E19. Our designed E19 molecule has the same tertiary structure as Ex4 but is more helical than Ex4 under all studied conditions; it is less prone to oligomerization and has preserved biological activity. These conditions make E19 a perfect lead compound for further drug discovery. We believe that this structural study improves our understanding of the relationship between local molecular features and global physicochemical properties such as water solubility and could help in the development of more potent Ex4 analogues with improved pharmacokinetic properties.
doi: 10.1021/bi500033c
This work was done during my PhD at the Eötvös Loránd University in Budapest, Hungary under the supervision of Prof. András Perczel.
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