Preliminary Structure And Dynamics Studies Of Membrane Proteins With Single Transmembrane Helix Using Solution Nuclear Magnetic Resonance

In this thesis, we used solution NMR to study the structure and backbone dynamics of membrane protein KCNE2and Integrin al trans-membrane and cytoplasmic domain (al-TMC). There are five chapters in this thesis.Chapter1is a brief review of membrane proteins and the new progress of structural and functional studies of membrane proteins. We introduced current structural investigating of membrane proteins using solution NMR, specially the opportunity and challenge for small membrane proteins. Chapter2is a brief review of the basic principles of NMR (Nuclear Magnet Resonance) and the methods and technology to resolve the structure of proteins using NMR.In Chapter3human helical membrane proteins have been predicted using TMHMM and SignalP methods with two databases:NCBI and Uniprot. The number of transmembrane helices, length distributions of transmembrane helices, and molecular weight of each membrane protein with a defined function were estimated from the predicted human helical membrane proteomes.In Chapter4we introduced the potassium channels contributing to the cardiac action potential. The voltage-gated potassium channel Ikr and Iks, the delayed rectifiers, are activated more slowly upon depolarization and return cells to rest during phase3repolarization; Ikr and Iks appearing to be carried by MiRP1/HERG and MinK/KCNQl channels, respectively, were discussed here. The β subunit of the Ikr and Iks Channel, the KCNEs were also discussed. Here, KCNE2was over-expressed, purified and eluted into different detergents for structural studies by solution NMR. It was not surprising that we found LMPG was the best detergent for KCNE2just like KCNE1whose structure was determined and reported earlier, since they both belong to KCNE family. We have determined the the NMR structure and analyzed the backbone relaxation of KCNE2. By comparing with the structure of KCNE1, many different structural details were found. The backbone dynamics data were consistent with the structure analysis.Chapter5discussed the integrin proteins family and their function for cell adhesion. The structural studies of transmembrane domain and cytoplasmic domain were focused on αⅡbβ3by NMR. We have over-expressed and purified the integrin α1-TMC and β1-TMC and determined the al-TMC structure. The TM-helix appears to form a kink at the position Phe62of TM-Helix. The TM-helix was spanning to the GFF of the GFFKR motif in the cytoplasmic domain. With the data of NMR titration by β1-TMC, interactions were suggested at the position of both sides of the boundary between TM domain and cytosol.