Structural, Dynamics And Functional Studies Of Ubiquitin Based On Paramagnetic Relaxation Enhancement

Ubiquitin (Ub) is a significant regulatory protein in eukaryote cells, and is involved in almost every physiological process. Ub interacts with myriad partner proteins to fulfill diversely functions; however, the interactions invariably involve a hydrophobic patch surrounding residue144. So how does Ub recognize a specific partner protein through such a conserved binding interface to regulate different cell signaling pathway?Furthermore, the complexity of Ub involved in physiological process is that Ub functions mostly as poly-ubiquitin chains. Two or more Ub molecules can be covalently linked via an isopeptide bond between the C-terminal carboxyl group of one Ub (dsital unit) and a primary amine of a lysine residue or N-teminal methionine in another one(distal unit). Lys63-lingked dimmer Ub (K63-diUb) is considered as a "typical" dimmer that is involved in cellular events like DNA damage response and innate cell immunity upon binding to a specific partner protein. Open conformation of ligand-free K63-diUb(apo K63-diUb) was characterized by many biophysical techniques; however, no closed conformation has been captured yet. Does apo K63-diUb exist in both open and closed states and whether there is a dynamic equilibrium between different states? Do these different conformational states could be interconverted? How does K63-diUb recognize and bind to a specific partner protein? Much more detailed descriptions of K63-diUb dynamics and conformational apace are highly deserved, and the connection between conformational dynamics and targets biding events should be addressed.Paramagnetic relaxation enhancement (PRE) affording long-range distance restraints favor protein structure determination. PRE is very sensitively to a distance change because of distance-dependence between paramagnetic center and protein nuclei, on a<r-6> magnitude. As a result, PRE could be used especially to characterize lowly-populated transient protein structures and ultra-weak protein-protein interactions.Here, we integrate PRE and other biophysical techniques and analysis methods to characterize the quaternary dynamics of Ub and K63-diUb. A rigid paramagnetic probe for PRE measurement is engineered to visualize subtle dynamics of Ub or other protein system. Preliminary dynamics and functional investigation of another important protein is also studied. In this study, we show that Ub dimerizes noncovalently through an interface around the β-sheet region with a KD of about5mM. The noncovalent dimmer interface overlaps with the conserved binding interface of Ub to target protein. Thus, the binding events between Ub and target protein probably could be regulated through the Ub monomer-dimmer dynamic equilibrium. The ensemble structures of Ub noncovalent dimmer adopt a wide range of relative subunit orientations, and some conformers are compatible with certain covalent linkage dimmer. As such, the covalent linkage in an Ub dimier may further promote noncovalent Ub-Ub interactions and favor a subset of the ensemble conformation, which would then tailor the interactions with a specific target protein.Based on the dynamics information of mono Ub, we resort to conjoined use of PRE and principal component analysis (PCA) to visualize conformational space of K63-diUb involved in target recognition. We show that K63-diUb adopts three conformational states, comprising one open state and two closed states, namely Cl and C2, with characteristic Boltzmann distribution—20%in open state,60%in Cl, and20%in C2. The three conformational states exist in dynamic equilibrium and can be readily interconverted. Importantly, each conformational state encompasses fhe known structures of K63-diUb with a particular ligand bound, and the respective binding affinity is weighted by the population of the preexisting conformational state. Thus, K63-diUb could recognize specific target protein through conformational selection mechanism based on the dynamics equilibrium and population shift between mutiple conformational states to regulate cell signaling and involved in different physiological process.In order to reveal subtler dynamics of Ub or other protein system, a rigid paramagnetic probe—diHis-Cu2+-cap is engineered. Attached with this rigid paramagnetic probe, PRE reveals that a flexible loop of Ub undergoes a collective pincer-like movement about2A on average, comprising residues52-53and55-56wobbled around residue54. In another protein system, holo glutamine binding protein (holo QBP), subtle dynamics (about0.6A on average) is also revealed with the rigid paramagnetic probe. This loop, comprising residue F13, packs against the glutamine ligand in the holo QBP. As such, the loop movement away from the ligand may facilitate the ligand dissociation.Last, PRE is also applied to reveal the desensitization mechanism of glutamate acceptor A2(GluA2). Preliminary results show that the desensitization rate is coupled with the dimeric strength of the ligand binding domain (LBD). Dimeric interactions stronger, GluA2desensitize slower; dimeric interactions weaker, then faster desensitization.In summary, this thesis work mainly characterize the dynamics of Ub and K63-diUb at quaternary level, and offer more understanding of protein function by detailed description of protein conformational space and protein dynamic equilibrium. Rigid paramagnetic labeling strategy and probe broaden the research content of PRE, and PRE will offer much more information in biological issues.