Conformational Dynamics Of Aquaporin Z And Diacylglycerol Kinase In The Phospholipid Membranes Revealed By Solid-state NMR

The functions of membrane proteins depend on the conformational rearrangements of specific active residues,and it is also closely related to the environment in which the membrane protein is located.Therefore,characterizing the conformational dynamics of membrane proteins with site-specific resolution in native-like environment is of great significance for understanding its functional mechanism.In this thesis,the site-specific conformational dynamics of aquaporin AqpZ and diacylglycerol kinase DAGK in phospholipid membrane were studied by taking advantage of solid-state Nuclear Magnetic Resonance(NMR)in studying membrane protein dynamics.The details are summarized as follows.Firstly,we compared the dynamics of different membrane proteins by observing the characteristics of solid-state NMR spectroscopy(resolution of NMR spectra,15N-13Cα cross polarization efficiency,NMR signal distributions based on dipole coupling and J-coupling,and the changes of signal intensity after hydrogen deuterium exchange)to provide basis and reference for the subsequent study of the site-specific dynamics.We found that the topological structures of membrane proteins in phospholipid membrane will affect the internal dynamics of proteins.The higher the proportion of transmembrane residues and the more transmembrane fragments,the better the conformational uniformity,the more rigid the structure,and the less significant the internal chemical exchange.Then,we investigated the dynamics of AqpZ with site-specific resolution using multidimensional solid-state NMR experiments to explore the significance of motions on the transport of water molecules across the membrane.We also explored the influence of the phase changes of E.coli native cell membranes and POPC/POPG synthetic phospholipid membranes on the dynamics of AqpZ.Firstly,we characterized site-specific 1H-15N,1H-13Cα,15N-13Cα dipolar order parameters(SNH2,SCαHα2,SNCα2)and rotating frame spin-lattice relaxation rate of 15N(15N-R1ρ)of the backbone of AqpZ in phospholipid membrane,and carried out theoretical dynamic model calculations.The results indicated that the transmembrane helix of AqpZ conducted small-amplitude(0-10°)collective motions on nanosecond time scales,which disturbs the pore size of the SF region and the hydrogen bond network in the water channel,thereby reducing the energy barrier for water molecule transport.Then,we measured the SNH2 and 1H-T1ρ(1/R1ρ)of AqpZ in different phospholipid phases.The results indicated that phase transitions of lipids in native membranes were less sensitive to temperature variations compared with that in synthetic bilayers,which resulted in that the dynamics of AqpZ were less affected by the phase transitions of lipids in native cell membranes,and the abundant membrane components of native cell membranes caused AqpZ to be slightly more dynamic at low temperature.Our research not only indicates the importance of the small-amplitude collective motions of the transmembrane helix of AqpZ for the water transport,but also demonstrates that native cell membranes are more conducive to helping membrane proteins resist the fluctuations of the environments and maintain stable dynamic behaviors.Lastly,we investigated the dynamics of DAGK with site-specific resolution using multidimensional solid-state NMR experiments to understand the significance of the conformational dynamics to its catalytic function.We characterized the site-specific SNH2,SCαHα2,SNCα2 and 15N-R1ρ of DAGK before and after binding to the substrate(ACP),and carried out theoretical dynamic model calculations.The results indicated that ACP significantly affected the motional amplitudes of DAGK’s local bond vector involved in the hydrogen bonding,but the collective motions of helix appearing in catalytic process already existed before ACP was bound.In summary,the local motions of DAGK are related to the specific selection of ACP,and the collective motions may play a role of "molecular lubrication" to promote the binding of ACP and the release of ADP.Our research increases the understanding of the catalytic mechanism of DAGK from the perspective of conformational dynamics.In summary,this thesis indicates that the topological structures of membrane proteins in phospholipid membranes will significantly affect the dynamics of membrane proteins;the collective motions of the transmembrane helix of AqpZ facilitate the water transport;the native cell membranes can help AqpZ resist the fluctuations of environment to maintain stable dynamic behaviors;the local motions and the collective motions of DAGK are important for the specific binding of substrates and the release of products.Our work has deepened the understanding of the importantance of motions in membrane protein functions.