| Abstract |
Nature provides peptides and proteins as a rich source of inspiration for the engineering of responsive, protein-based nanomaterials for medical and biotechnological applications. The function of proteins is strongly dependent on their structures. Despite scientific progress in this field, understanding how proteins dictate their unique three-dimensional structures and physicochemical properties remains an unsolved puzzle. A detailed understanding of sequence-structure relations in proteins would provide us with the key insights to develop novel bio-inspired materials with desired functionalities.
Among the most abundant structural motifs found in proteins, the α-helical coiled coils serve as useful models for analyzing protein folding and understanding the relationships between sequence and structure, as they combine structural simplicity, remarkable functional versality, and structural plasticity in a wide variety of topologies and folding states. Nowadays, several mathematical and computational techniques play an important role in investigating proteins in aqueous solutions.
Initially, this study investigates the folding and thermal stability of two well-characterized, highly regular four-α-helical bundle proteins: the repressor of primer (Rop) protein and its loopless mutation RM6, using all-atom molecular dynamics (MD) simulations. A detailed examination of the structural and conformational properties of wild-type Rop (wtRop) and RM6 reveal different physical characteristics even in their native states. Our findings reveal that RM6 exhibits greater thermostability than wtRop on various measures. Deviations from native structures are detected mostly in tail and loop regions and most flexible residues are indicated. A decrease in hydrogen bonds with the increase of temperature is observed, as well as a reduction in the number of hydrophobic contacts in both proteins. The primary aim of this study is to explore at the atomic level how a protein mutation can cause major changes in its physical properties, such as its structural stability.
Subsequently, critical knowledge about the sequence structure relationships comes from the investigation of retro proteins, where the amino acid sequence of a known protein is reversed. Expanding our research, we study the sequence amino acid reversal of the wtRop via all-atom MD simulations. MD simulations were performed on two different types of reversed protein models of Rop, one with a completely reversed amino acid sequence (rRop) and another with a partially reversed sequence (prRop), where the five residues of the loop region (30ASP-34GLN) were not reversed. The exploration of the structure of the two retro models is performed, highlighting similarities and the differences with their parent protein, by employing various measures. The simulation findings reveal a disruption of the α-helical structure of both retro proteins, rRop being more stable than prRop. A corruption of their hydrophobic core is also observed. Two models have been studied for both reversed proteins, a dimeric and a monomeric, and the former to be more stable than the latter. The initial structures of both dimer models had an antiparallel orientation between the monomers, similar to that of the wtRop protein.
In the final part, the focus of this thesis shifts to the totally reversed sequence of the amino acids of wtRop protein. Given the absence of a known native state for rRop, we employ AlphaFold as an alternative approach to generate its initial structure. Therefore, all-atom MD simulation of one of the generated rRop models with a parallel orientation between the monomers in aqueous solution was performed. Our analysis, using a variety of measurements, highlights the similarities and differences between rRop and the wtRop protein. Additionally, due to the limitations of classical MD simulations, two different enhanced sampling techniques were also utilized that are the well-tempered metadynamics, using various collective variables and the replica exchange molecular dynamics. The simulation results reveal a disruption in the α-helix conformation of the rRop protein, along with a decrease in its conformational stability compared to the wtRop protein. Furthermore, rRop exhibits a preference for a twisted orientation between the two monomers. Finally, through the computation of the transition temperature of wtRop and different rRop models no significant effect was revealed through the reversal of the amino acid sequence.
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