| Proteins perform their functions mainly through protein-protein interactions (PPIs), and different types of PPIs taken in living cells carry out different molecualr processes. Traditionally, dissociation constant (KD) value is used to describe the binding affinity of PPIs,and PPIs can be classified into two categories based on the dissocation constant, which are stable interaction and transient interaction. The KD value of stable interactions span from picomolar (pM) to micromolar (μM), and other PPIs with the affinity value from micromolar to millimolar are defined as transient interactions. Basically PPIs with KD value over 1 μM are also categorized as weak protein-protein interactions. Traditionally, stable Protein-Protein Interactions can be characterized not only for their structures, but also their specific functions with X-ray, EM-Cryo, NMR and other biochemical and biophysical techniques. While as the restriction of techniques, structures and fuctions of weak inter-protein interactions including transient PPIs are not easy to be characterized. What is meant by this is that the structures and functions of weak PPIs are still await to be identified and illustrated.In the study of structure biology, the technique named solution nuclear nagmetic resonance (NMR) spectroscopy of proteins is widely used, as it can be applied to obtain information about the atomic resolution structure and dynamics of proteins and PPIs complex in solution. The information contains lots of experimentally deter mined restraints, such as distances restraints, angle restraints and orientation restraints. As protein samples for NMR experiments were dissolved in buffer, it is extremely fantastic to use NMR techniques to visualize transient or ultraweak PPIs distinguised with X-ray scattering and EM-Cryo.Here, we applied modern solution NMR spectroscopy methodologies complemented with many other biophysical and biochemical methods to characterize the structure and function of three sorts of PPIs.The first type PPIs is ultraweak inter-protein interactions.Our researches firstly visualized an ultraweak protein-protein interaction with an affinity of approximate 25 millimiloar during phosphorylation signaling with the technique named paramagnetic relaxation enhancement (PRE). Which means a phosphoryl group can still be transferred from one protein to the other by interacting with each other with an affinity of only about 25 millimolar. We deter mined the atomic resolution structure of the complex using distance restraints from state-of-the-art paramagnetic NMR technique, illustrated the physical basis of this kind of weak PPIs:compromise with hydrophobic effect and electrostatic repulsion effect, estimated the life-time of the complex by molecular dynamic simulation. Based on mathematical modelling, we proposed that ultra-weak (fleeting) interaction enforced rapid speed of phosphoryl signal transfering when providing a high effective protein concentration. This work may pave the way to investigate the vast reserve of fleeting interactons between proteins.The second type PPIs is transient homo-dimer inter-protein interactions. Our another research focuses on illustrating the structure, allostery and function of the dimerized C-ter minus domain of capsid protein during the process of HIV-1 capsid remodeling. We proved that two strictly conserved cysteine residues in the capsid protein C-ter minal domain (CAC) can be oxidized from in-cell to out-cell by cyclic voltammetry and NMR. Moreover, the disulfide bond formation triggers dramatic orientational change of the dimerized structure of capsid protein C-ter minus domain based on the structure we calculated. The shape information of reduced and oxidized capsid protein homo-dimer was acquired respectively against SAXS based restraints, and the difference between them was close and open, which perfectly agrees with the structures obtained by EM-Cryo. Further, our co-worker’s results demonstrated that new born virions formed under reducing condition, that prevent CA oxidation, exhibited abnormal core morphology and were non-infectious. In conclusion, our work showed that oxidation induced allosterical change of capsid protein C-ter minus domain homo-dimer interaction may be one of the factors triggering large-scale remodeling of the capsid structure, we may revealed a potential mechanism of capsid remodeling in HIV-1 maturation.The third type PPIs is transient homo-oligomer inter-protein interactions. Solution NMR techniques were carried out to reveal the structure and function of HIV-1 SP1 domain homo-oligomer, SP1 domain contains 13 a mino acids and is cut off from the C-ter minus domain of capsid protein during the process of HIV-1 maturation. Preli minary results show that the secondary structure of SP1 domain was random coil and the SP1 domain formed homodimer in solution with an affinity value of 0.67±0.16mM. We also proved that the dimerization of SP1 domain could not enhance the dimerization of the C-ter minus domain of capsid protein, while the dimerization of the C-ter minus domain of capsid protein could weaken the dimerization of SP1 domain.In summary, this dissertation contains using solution NMR to characterize the structure and function of three kinds of PPIs complex, we firstly developed a series of techniques to study the structure and dynamics of ultraweak PPIs in phoshorylation signaling, discovered the probable molecular mechanism of capsid structure rebuliding during the process of HIV-1 maturation, initially demonstrated the inter-protein interactions of HIV-1 SP1 homo-oligomer. This work enriched researches on weak protein-protein interactions, and provided the basis for researches on the structure and function of weak protein-protein interactions. |
