Design and characterization of Trp-cage miniproteins
Miniproteins are adequate models to study various protein-structure modifying parameters, such as temperature, pH, point mutations, H-bonds, salt bridges, molecular packing, etc. Tc5b, a 20-residue Trp-cage protein is one of the smallest of such models with a stable 3D fold. However, Tc5b exhibits considerable heat-sensitivity and is only stable at relatively low temperatures. We reported a systematic investigation of structural factors influencing the stability of Tc5b by solving its solution structure in different environments, varying temperature, and pH. Selected variants (mutated, glycosylated and truncated) of Tc5b were designed, prepared, and investigated by NMR. We designed a new variant, Tc6b, differing only by a methylene group from Tc5b, in which both key interactions are optimized simultaneously. Tc6b exhibits enhanced heat stability and adopts a stable fold at physiological temperature.
Mapping the key residue-residue contacts responsible for Trp-cage stability, designing variants and investigating them by NMR- and CD-spectroscopy showed that the 9-16 salt bridge is integrated into the cooperativity network of the molecule rather than being an isolated stabilizing factor. Furthermore, our results indicated that the folding of Trp-cage miniproteins is more complex than a simple two-state process as 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 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. Heteronuclear relaxation studies combined with MD simulations revealed the source of backbone mobility and the nature of structural rearrangements during these transitions.
We also characterized the amyloid formation of several sequence modified and side-chain phosphorylated Trp-cage variants applying NMR, CD and FTIR spectroscopies, MD simulations, and TEM in conjunction with ThT fluorescence measurements to reveal the structural consequences of side-chain phosphorylation. We demonstrated that the native fold is destabilized upon serine phosphorylation, and the resultant highly dynamic structures form amyloid-like ordered aggregates with high intermolecular β-structure content. We proposed a complex aggregation model for these Trp-cage miniproteins.
The ability to detect structural and dynamic information about folding intermediates in vitro provides an excellent opportunity to gain new insights into the energetic aspects of the energy landscape of protein folding and Trp-cage miniproteins are indeed a realistic model of larger globular systems of composite folding and aggregation landscapes and helps us to understand the fundamentals of deleterious protein aggregation and amyloid formation.
A Trp-cage miniprotein with key stabilizing residues highlighted
Dr. Gábor Tóth, Department of Medicinal Chemistry, University of Szeged, Hungary,
Dr. László Nyitray, Department of Biochemistry, Eötvös Loránd University, Budapest
József Kardos , Bence Kiss , András Micsonai , Petra Rovó , Dóra K. Menyhárd , János Kovács , Gábor K. Tóth , András Perczel
Phosphorylation as Conformational Switch from the Native to Amyloid State: Trp-Cage as a Protein Aggregation Model
J. Phys. Chem. B 119(7):2946–2955. | DOI: 10.1021/jp5124234 | PMID: 25625571 (2015) Kivonat