Functional and structural studies of membrane transporters
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- Tez No: 611930
- Danışmanlar: PROF. DR. DANIŞMAN YOK
- Tez Türü: Doktora
- Konular: Biyokimya, Moleküler Tıp, Biochemistry, Molecular Medicine
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2018
- Dil: İngilizce
- Üniversite: Eidgenössische Technische Hochschule Zürich (ETH)
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 109
Özet
The structural information on membrane proteins (MPs) is critical to understand the molecular mechanism of ligand recognition, selectivity and transport. For structural studies and many other purposes, MPs need to be extracted by the help of detergents from their native environment, the lipid bilayer. The literature about the amounts of detergent and endogenous phospholipid molecules bound to MPs after purification was limited. In order to have a better understanding on this concept, we carried out a systematic study with three model integral MPs of different oligomeric states purified in nine different commonly used detergents. The insights gained from this study can be summarized as follows. 1) Purified integral MPs are ternary complexes consisting of protein, lipid, and detergent. 2) The amount of detergent and endogenous phospholipids bound to purified proteins are dependent on the size of the hydrophobic lipidaccessible protein surface areas and the physicochemical properties of the detergents used. 3) The size of the detergent-lipid belt surrounding the hydrophobic lipid-accessible surface of purified MPs can be tuned by appropriate choice of detergent. 4) The detergents n-nonyl-β-Dglucopyranoside (NG) and 5-cyclohexyl-1-pentyl-β-D-maltoside (Cymal-5) showed exceptional delipidating effects on the model proteins during purification. 5) The types of endogenous phospholipids bound to MPs could vary depending on the detergent used for solubilization and purification. 6) Size-exclusion chromatography (SEC) was shown to be a reliable method for estimating the molecular mass of ternary complexes of integral MPs. These findings propose a strategy to control and tune the number of detergent and endogenous phospholipid molecules bound to MPs. Importantly, control of the composition of these ternary complexes is fundamental for the successful crystallization of MPs and consequently for MP structure determination by crystallographic approaches. By applying this strategy we could minimize the size of the detergent-lipid belt around the L-arginine (Arg)/agmatine (Agm) antiporter AdiC from Escherichia coli (E. coli) and make the formation of crystal contacts between proteins sterically possible. As a result, we could crystallize and solve the crystal structure of the wild-type (wt) form of AdiC at the unprecedented resolution of 2.2 Å in the substrate-free outward-open conformation, and for the first time with the substrate Agm at 2.6 Å resolution in the same conformation of the translocation cycle. The Agm-bound crystal structure indicated that Agm binds to the same binding pocket as Arg in the previously published outward-facing occluded conformation. The identified subtle differences between the Arg- and Agm-bound AdiC structures describe for the first time the different affinities of this transporter at the molecular level. In the light of our findings and the previously published AdiC structures, we could describe the conformational changes of AdiC connected with the release of Agm into the periplasmic space
Özet (Çeviri)
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