Interactions Of Three Types Of Transmembrane Protein With Lipids Investigated By Solid-state NMR

Membrane proteins play key roles in many important biological processes,such as substrate transport,signal transduction and energy conversion.Many integral membrane proteins are modulated structurally and functionally by the surrounding lipids.In this case,the effects of lipids on membrane proteins can be divided into two categories:firstly,indirect effects on the structure and function of membrane proteins by the physical properties of lipid membranes,including thickness,curvature,asymmetry,and fluidity.Secondly,direct effects on the folding,assembly and function of membrane proteins by specific interactions with endogenous bound lipids.Solid-state nuclear magnetic resonance(NMR)has unique advantages in studying membrane protein-lipid interactions in a lipid membrane environment.As protein-lipid interactions vary considerably in different types of membrane proteins,three typical types of membrane proteins were studied for this thesis.The single-transmembrane protein synaptobrevin-2(Syb-2),the multi-transmembrane mechanosensitive ion channel of large conductance(MscL)and the transporter SemiSWEET(Sugars Will Eventually be Exported Transporter)were selected to systematically investigate the two types of interactions between membrane proteins and lipids.The specific studies are as follows:1.Indirect effects of the physical properties of lipid membranes on the structure,dynamics and function of membrane proteins.1)The effect of the lipid membrane environment on the structure and dynamics of the single transmembrane protein Syb-2.Many studies have found that differences in the membrane mimetic environment have an impact on the structure and dynamics of the simple single transmembrane protein Syb-2.Complexes I,II and III in the lipid membrane environment were constructed to mimic the three assembly stages in the SNARE-mediated membrane fusion process,and the conformational and dynamic changes of Syb-2 in these three complexes were characterized using solid-state NMR.The results show that the structure and dynamics of the transmembrane region of Syb-2 in lipid bilayers differs from its crystal structure in the detergent environment.The transmembrane region of Syb-2 in the lipid bilayers did not form a stable helical bundle structure with syntaxin-1 as that in the detergent,thereby providing new insights into the structural mechanism of SNARE complex assembly and highlighting the importance of the membrane environment in elucidating the functional mechanism of membrane proteins.2)The effect of the lipid membrane environment on the structure,dynamics and function of the multi-transmembrane MscL.MscL is a class of lipid-gated channels that are sensitive to changes in the lipid membrane environment.The topology of the Ma-MscL(MscL from Methanosarcina acetivorans)in artificial lipid membranes and native cell membranes was characterized using solid-state NMR,showing that the structure of local regions of the Ma-MscL were perturbed by different membrane-mimicking environments.Based on the site-specific measurements of the 15N-1H,13Cα-1H and 13Cα-15N dipolar order parameters and 15N rotating frame spin-lattice relaxation rates(R1ρ),it was found that both the well-defined transmembrane regions and the less structured intramembrane loops undergo restricted submicrosecond time scale motions to maintain the closure of the channel.In addition,the hydrophobic gating mechanism of the Ma-MscL in the lipid membrane environment was revealed by using solid-state NMR H/D exchange and water-edited experiments,and functional assays.This study explores the structural,dynamic and functional mechanisms of the Ma-MscL in the lipid membrane environment by means of various solid-state NMR experiments,providing a paradigm of the effects of the lipid membrane environment on the structure,dynamics and function of multiple transmembrane channel.3)The effect of lipid membrane environment on the structure and dynamics of the multi-transmembrane transporter Vibrio sp.semiSWEET(Vs-SemiSWEET).By comparing the chemical shifts and signal intensities of Vs-SemiSWEET in lipid membrane environments of different thicknesses and charges,it was found that both the thickness and charge distribution of membranes affect the structure and dynamics of the membrane interface and the inter-monomer interface regions.Further,the three-dimensional structure of the Vs-SemiSWEET in lipid bilayers was obtained for the first time based on a large number of experimentally determined interatomic distances and local torsional restraints.The structure is significantly different from the crystal structure of that in the lipid cubic phase,thus further emphasizing the influence of the membrane environment on the structure of the multi-transmembrane transporter.2.Specific interactions between endogenous bound lipids and membrane proteins.The interactions between endogenous bound lipids and Vs-SemiSWEET were analyzed in detail from multiple perspectives using solution NMR,solid-state NMR,biochemical experiments and molecular docking methods.The types and numbers of endogenous bound lipids in different species of SemiSWEET were characterized by solution NMR 31P spectra,and all species of SemiSWEET were found to have endogenous bound PE(Phosphatidyl ethanolamine),PG(Phosphatidylglycerol)and CDL(cardiolipin)molecules.The binding sites of endogenous lipids were confirmed by the 13C-13C spectra of Vs-SemiSWEET with long mixing time,and the results showed that lipid molecules were mainly distributed in two regions:the transmembrane pores and the inter-monomer interface.The binding sites of lipids in the inter-monomer interface region were further mutated and the effects of different types of lipids on the denatured Vs-SemiSWEET were compared.The presence of endogenous binding phospholipids was found to promote the formation of hydrogen bonds between monomers,thus revealing the molecular mechanism of endogenous bound lipids regulating the oligomerization of Vs-SemiSWEET.In summary,this thesis used solid-state NMR to explore the indirect effects of lipid membrane environment on the structure,dynamics,and function of different types of membrane proteins systematically.For the simple single transmembrane protein Syb-2,only inter-residue hydrogen bonds within the α-helix are present in the transmembrane region to maintain structural stability,and there are no interactions between transmembrane structural domains,making it more susceptible to the surrounding environment.In contrast,for the multi-transmembrane multimeric proteins Ma-MscL and Vs-SemiSWEET,both of which have multiple α-helical transmembrane structural domains.In their transmembrane regions,there are interactions not only between α-helical transmembrane structural domains within the monomer but also between monomers,thus the membrane-mimicking environment does not have a significant effect on the overall structure of the Ma-MscL and Vs-SemiSWEET.However,the local structures of the two proteins,especially those in the near-membrane regions,are affected by the membrane-mimicking environment,leading to significant differences in the structure-based molecular mechanisms.On the other hand,the direct effects of endogenous bound lipids on membrane protein structure were explored by solid-state NMR,revealing the molecular mechanism by which endogenous bound lipids regulate Vs-SemiSWEET protein dimerization.This paper provides new insights into transmembrane protein-lipid interactions,demonstrates the necessity of studying membrane protein structure and functional mechanisms in a lipid membrane environment,and demonstrates the unique advantages of solid-state NMR in membrane protein-related studies.