Thermodynamic and structural analysis of ultrafast folding proteins

Resumen

This Doctoral Thesis studies different fast folding proteins through diverse thermodynamic techniques and structural methods, to obtain cooperativity and behaviour information. Among the thermodynamic techniques are different equilibrium spectrometry techniques as far-UV Circular Dichroism, Fluorescence, Fourier Transform Infra-Red, as well as Differential Scanning Calorimetry. The protein candidates are expected to have fast folding, with folding under the microseconds timescale. Until mid 1990s fast folding proteins were out of the available techniques until the breakthrough of different techniques that allowed working with faster timescales. Fast folding proteins proteins expand the knowledge of the folding and might cover the gap between experimental and computational methods, since traditionally the last ones have been able to explore only processes on the sub-millisecond timescale. The experimental identification of the different folding scenarios predicted by the folding funnel hypothesis of the energy landscape theory by Bryngelson et al. [1] is a fundamental step. In 1999 V. Muñoz and W.A. Eaton [2,3] proposed the selection of some candidates to search for downhill folding scenarios versus the classical two-state folding mechanism. Fast folding proteins frequently show downhill folding since a small or inexistent energy barrier speeds up the folding process, and they may function as molecular rheostats [4-6], with different functions depending on the conformation. For this Thesis project we focus on possible candidates of the downhill folding mechanism since the shallow energy barrier allows to study the barrier top populations [7]. Therefore, initially two relatively small proteins with different secondary structure content were selected to study the folding patterns related to these structures: a WW domain (the third domain from mouse Ubiquitin-protein ligase Nedd4-2), with all beta-sheets; and a R3H domain (from the human sperm-associated antigen 7), with beta-sheet and alpha-helix secondary structure. These domains showed different thermodynamic behaviour related to their secondary structure content and hydrophobic forces among others. The thermodynamic study of the WW domain shows features typical of the downhill-like scenario, with structural flexibility in the native state and broad unfolding transitions and melting temperatures dispersion. However, the R3H domain has a cooperative unfolding despite the slight loss of structure in the pre-transition region by the fraying of the alpha-helices. Furthermore, two interesting proteins from a folding point of view are also studied with different techniques in the field of the Nuclear Magnetic Resonance (NMR), the HP35 subdomain from Villin protein, and gpW protein. The HP35 subdomain has been frequently used for molecular simulation studies, and we will focus on the atomic unfolding of some residues with NMR taking as reference the atom-by-atom NMR study by Sadqi et al. [8] on the one-state downhill folder BBL domain (from the E2 subunit of the 2-oxoglutarate-dehydrogenase enzyme from Escherichia coli). This subdomain presented less heterogeneity than would be expected for a fast-folding protein (which folding would be initially predicted towards the downhill regime), and becomes the fastest protein with two-state folding. A similar study was performed by Sborgi et al. [9] on gpW protein, for which the atom-by-atom shows the behaviour of different NMR probes (amide 15N and 1HN, 13Cα and 13Cβ), which can be complex if the process is progressive like for downhill proteins [8] or more homogeneous like for two-state proteins [10] (even different tendencies may appear along the temperature range). Hence, the thermal unfolding of gpW protein has been thermodynamically studied [11], along with analysis of the process at atomic level by NMR [9]. However, its native state may be a non-static structure since proteins change upon variations in their environment. To be able to deepen our understanding of the native state of gpW protein there was a barrier to overcome, since it has a folding rate too fast for the timescale affordable by Relaxation Dispersion NMR. This technique distinguishes meta-stable conformations at levels almost undetectable with regular techniques, that usually average structures, making impossible to discern this very low populated states. Our detailed analysis provides information on the exchange between the native and the low populated, “invisible”, state, reflecting the non-static protein structure, in which this last state has almost intact alpha-helices while the beta-hairpin is unfolded, even at low temperature, and appears as a productive intermediate at the end of the kinetic folding pathway. This low stability region of the beta-hairpin was observed in other systems, and has been proposed to play a possible role as conformational switch [12]. 1 Bryngelson, J. D., Onuchic, J. N., Socci, N. D. & Wolynes, P. G. Funnels, Pathways, and the Energy Landscape of Protein-Folding: A Synthesis Proteins-Structure Function and Genetics 21, 167-195 (1995). 2 Munoz, V. & Eaton, W. A. A simple model for calculating the kinetics of protein folding from three-dimensional structures. Proceedings of the National Academy of Sciences of the United States of America 96, 11311-11316 (1999). 3 Eaton, W. A. Searching for "downhill scenarios" in protein folding. Proceedings of the National Academy of Sciences 96, 5897-5899, doi:10.1073/pnas.96.11.5897 (1999). 4 Garcia-Mira, M. M., Sadqi, M., Fischer, N., Sanchez-Ruiz, J. M. & Munoz, V. Experimental identification of downhill protein folding. Science 298, 2191-2195 (2002). 5 Li, P., Oliva, F. Y., Naganathan, A. N. & Munoz, V. Dynamics of one-state downhill protein folding. Proc Natl Acad Sci U S A 106, 103-108 (2009). 6 Muñoz, V., Campos, L. A. & Sadqi, M. Limited cooperativity in protein folding. Current Opinion in Structural Biology 36, 58-66 (2016). 7 Gruebele, M. in Protein Folding, Misfolding and Aggregation 106-138 (RSC Publishing: London, 2008). 8 Sadqi, M., Fushman, D. & Munoz, V. Atom-by-atom analysis of global downhill protein folding. Nature 442, 317-321, doi:10.1038/nature04859 (2006). 9 Sborgi, L. et al. Interaction networks in protein folding via atomic-resolution experiments and long-time-scale molecular dynamics simulations. J. Am. Chem. Soc. 137, 6506-6516 (2015). 10 Campos, L. A. et al. Gradual Disordering of the Native State on a Slow Two-State Folding Protein Monitored by Single-Molecule Fluorescence Spectroscopy and NMR. The Journal of Physical Chemistry B, doi:10.1021/jp403051k (2013). 11 Fung, A., Li, P., Godoy-Ruiz, R., Sanchez-Ruiz, J. M. & Munoz, V. Expanding the realm of ultrafast protein folding: gpW, a midsize natural single-domain with alpha+beta topology that folds downhill. J. Am. Chem. Soc. 130, 7489-7495, doi:10.1021/jaB01401a (2008). 12 Balusek, C. et al. in Quantitative Models for Microscopic to Macroscopic Biological Macromolecules and Tissues 1-20 (Springer, 2018).