Research To The Thermal Stability, Folding And Intermediate Ensemble Of HUBF HMG Box-5 Using Nuclear Magnetic Resonance (NMR) And Biophysical Spectroscopy

Understanding the details of how the hierarchy of interactions directs the protein structural stability and the folding process remains one of the fundamental questions in biology. Answers to this question require investigations of the folding process and equilibrium intermediates using different techniques. Some model proteins show a two-state folding equilibrium with a cooperative change of different structural elements. Many other proteins, however, display more complex folding equilibrium in which stable intermediates accumulate. Recently, much important progress has been made through detailed structural and dynamic studies of equilibrium intermediates, and it is generally accepted that equilibrium intermediates produced in vitro resemble the kinetic counterparts and non-native conformations produced in vivo.Our work focuses on the following two parts: (1) Detailed investigation into the reversible three-state thermal unfolding of Box-5 using NMR, DSC, CD spectroscopy, Fluorescence spectroscopy and Stopped-Flow experiments. We found a heavy-populated thermal intermediate ensemble of Box-5 at 55℃for the first time. Then we characterize the conformation and backbone dynamics of this intermediate ensemble using those techniques mentioned above. Detailed NMR relaxation dynamics are compared between the native state and the intermediate ensemble. Our results implicate a fluid helix-turn-helix folding model of Box-5, where helices 1 and 2 potentially form the helix 1-turn-helix 2 motif in the intermediate, while helix 3 is consolidated only as two hydrophobic cores to stabilize the native structure. The fluid helix-turn-helix folding model is proposed for the first time in the HMG boxes family. (2) The clone, expression, purification and structural and interaction with a 14bp dsDNA of Sox-4 HMG domain.Chapter 1 provides a brief review of protein stability and folding in vitro, which includes the traditional theory of protein stability and folding. A detail introduction about new idea of folding mechanisms and species related with protein folding is the key point of this section. In the last paragraph, we introduce some new examples which have focused on the relationship of protein stability, folding and function.Chapter 2 provides a detailed review about various experiments for the study of protein folding and stability. Most is related with the methods using in this dissertation, e.g., DSC, biophysical spectroscopy, Stopped-Flow and NMR. A detail introduction about NMR study of non-native state of protein is the key point of this section, which includes various related NMR parameters (CSI, NOE, PRE, and RDC) and relaxation spectral density mapping method.Chapter 3 is the key point of this dissertation, which includes the experimental results and discussion about the stability and folding study of Box-5. DSC data show that Box-5 unfolds reversibly in two separate stages. The first transition (N←→I) accounts for 30% of the total unfolding enthalpy, and the second one (I←→U) for 70%. Spectroscopic analyses suggest that different structural elements exhibit non-cooperative transitions during the unfolding process and that the major form of the Box-5 thermal intermediate ensemble at 55℃shows partially unfolded characteristics. Compared with previous thermal stability studies of other boxes, it appears that Box-5 possesses a more stable major wing and two well separated sub-domains. NMR chemical shift index and sequential ¹H^N_i -¹H_Ni+1 NOE analyses indicate that helices 1 and 2 are native-like in the thermal intermediate ensemble, while helix 3 is partially unfolded. The conformation of this thermal intermediate ensemble is heterogeneous, with about 12.8% of the signal contributed by the native state and about 0.5% from the unfolded state. Backbone ¹⁵N relaxation parameters show that the native state of Box-5 exhibits a large difference in the correlation times between the overall tumbling (9.5ns) and the internal motions (1.1ns). In comparison, in the Box-5 intermediate ensemble, less inhomogeneous NH mobility throughout the backbone is indicated, with a faster overall tumbling (4.7ns) and similar internal motions (1.3ns). The data of NMR conformational entropy change between native state and intermediate show that W14, W41, W52 and Y63 have largest increase of conformation entropy, which indicate critical importance of those residues for the conformational stability of native state. However, residues of V18, K27, A56 and the c-terminal residues show negative conformation entropy, implicating some non-native interaction involved around these residues in conformation of intermediate ensemble. In addition, the kinetic intermediate of Box-5 was also detected by the stopped-flow GdnHCl experiments. In total, our results implicate the folding mechanism of hUBF HMG Box-5, where helices 1 and 2 are pioneer species and are potentially packed as a fluid helix-turn-helix (HTH) motif, whereas helix 3 is just partially formed in the intermediate. Further work is needed to confirm our hypothesis about fluid HTH folding model of Box-5 in the future.Chapter 4 is talk about the clone, expression, purification and structural and interaction with a 14bp dsDNA of Sox-4 HMG domain. The full name of Sox is SRY (Sex-determining Region, Y chromosome) -related box. Our results of gel shift assay proof the sequence-specific interaction of DNA (5'-AACAAAG-3') with Sox-4 HMG domain. ITC results show that the interaction of DNA and Sox-4 HMG is entropy-droved, and the dissociation constant is about 1.30*10^-6M. NMR chemical shift perturbation results indicate that some charged residues, e.g., H27, R38, H61 and R56, potentially involved in the interactive site of Sox-4 HMG protein with DNA. NMR relaxation data indicate that the motion in region of helix 1 and helix 2 is significantly restrained in the DNA complex state of Sox-4 HMG protein. However, compared with the total entropy change from ITC experiments, the value of conformational entropy change derived form NMR relaxation data is only about 1/14 of total entropy. It is suggested that the entropy-droved interaction of Sox-4 HMG and DNA is less related with the backbone conformational change.