The role of structural dynamics in protein function and evolvability.

During evolution 'peptides' are turned into 'proteins' by delegating the specific functions to such biopolymers in a sophisticated manner. Out of all available conformers, nature has to select a subset of them to allow each protein to recognize specific substrates. During the short evolutionary period, such functional specialization of a fix-length peptide can be faithfully explained by the protein evolvability theory. To rationalize the evolution of the non-fixed length peptides that might occur upon a long-term period, we focused on expanding the current evolvability theory by introducing the protein modularity notion in this thesis.

To fully comprehend the concept of protein modularity, we provided an overview in Chapter 1 that includes the summary of two fundamental concepts: (i) the multi-tier protein dynamics and (ii) the protein evolvability. The chapter also includes a short explanation of the energetic funnel model, as it allows us to hypothesize the evolution of the protein energetics and structures upon functional specialization. By elaborating on those widely-accepted concepts, we are able to form a working hypothesis on the role of the protein modules, as we presented in the subsequent Chapter 2.

Chapter 2 provides a generic working hypothesis of this thesis by questioning the modules' role to specialize the function of proteins during evolution. To verify this hypothesis, we investigated a large group of proteins adopting a common 'primordial' structure, i.e., the 'cherry-core' (CC). The cherry-core idealized consensus, which is composed of two continuous Rossmann-fold topology domains (D1 & D2) connected via two anti-parallel β-strand 'hinges' (βH1 & βH2), exhibits a remarkable symmetry which may contribute to its conformational variability and plasticity. We then proposed that the modules' geometrical placement (i.e., C-terminal additions) in the CC enables the multi-tier dynamics on the CCPs.

To test the hypothesis stated in the previous chapter, we probed large-scale domain motions via single-molecule Förster Resonance Energy Transfer (smFRET) and local structural fluctuation via Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) of the CCPs and their derivatives. In Chapter 3, we found that the modules of CCPs confer distinct multi-tier structural dynamics to the CC. Such dynamics allow the diversification of function and ligand specificity of the CCPs. In this chapter, we focused on highlighting the role of the asymmetric C-tails to 'generate' tier-0 dynamics on SBD2 and MalE of Class B and G, respectively. Such proteins are ABC transporter-related proteins, which require two distinct conformations to capture their cognate ligands (i.e., open in the apo- and closed in the holo-state). By disrupting specific interactions on the C-tail, we destabilize the open apostate and increase their binding affinity. Such results confirm the role of the modules of the ABC transporter-related CCPs to generate the 'open' conformation as the native apo-state.

The cherry-core of the LysR Type Transcription Regulators (LTTRs) is having no additional C-tail module and acting as the effector binding domain (EBD). We showed that Tier-0 dynamics are not observable on the EBD of CynR and may not be required during effector binding to propagate the quaternary changes. In Chapter 4, we monitored the lower tier dynamics that underlie the function of CynR by using the combination of structural modeling and dynamics studies (i.e., HDX-MS). While there are no significant structural changes observed on the CC detected with our experimental procedures, we proposed that the accumulative low tiers dynamics are possibly sufficient to initiate the quaternary changes essential for the DNA-(un)bending.

In Chapter 5, we determined the modules' effect on the structural dynamics of one of the modern CCPs, i.e., the Maltose Binding Protein (MalE). Such modules are embedded predominantly at the C- terminy and integrated within the conserved structural more as the substitutes of consensus helices/sheets, as detailed in Chapter 2. We verified the role of such modules using an array of biophysical tools (i.e., smFRET, HDX-MS, and Isothermal Titration Calorimetry) and MD simulations that allowed us to reconstruct the energetic landscape of MalE. By altering such modules using site-directed mutagenesis, we confirmed the modules' synergistic roles, which are critical to personify MalE to function as a transport-related binding protein by (i) entrapping the ligand and (ii) creating a distinct apo-state.

We anticipated that the energetic arguments might be seen as the main selective pressure during evolution. To fulfill the energetic requirement, nature might introduce modules into the highly-evolvable protein cores during the long-period evolution, as we presented on the modularity concept in this thesis. Once the modules are 'attached,' evolution on a fix-length peptide is sufficient to optimize the protein functions. The later evolutionary process might take effect in a relatively shorter evolutionary-period, as stated in the ‘avant-garde’ evolvability theory.