Purification and functional characterization of nine human Aquaporins produced in Saccharomyces cerevisiae for the purpose of biophysical characterization

The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized, cheap and easily handled human membrane protein production platform, we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs, focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purification of histidine tagged human AQP10 produced in large bioreactors. Glycosylation analysis revealed that AQP7 and 12 were O-glycosylated, AQP10 was N-glycosylated while the other AQPs were not glycosylated. We furthermore performed functional characterization and found that AQP 2, 6 and 8 allowed flux of water whereas AQP3, 7, 9, 10, 11 and 12 also facilitated a glycerol flux. In conclusion, our S. cerevisiae platform emerges as a powerful tool for isolation of functional, difficult-To-express human membrane proteins suitable for biophysical characterization

Generation and exploitation of proton motive force: Biochemical and structural analysis of three bacterial integral membrane proteins.

Proton motive force, a necessity for all living cells, is generated and exploited by asignificant number of membrane-bound enzymes and transporters. The focus of thisthesis was to understand the physiological relevance, structure and mechanism of threesuch bacterial enzymes: proteorhodopsin; transhydrogenase; and AcrB.Proteorhodopsin, common in surface living bacteria, is demonstrated to exist also inphylum Bacteriodetes, the third most abundant class of ocean bacteria. Evidence is alsopresented that its presence stimulates growth of one such bacterium in the presence oflight. This finding indicates that proteorhodopsin associated growth enhancementcould contribute to the primary production in earth’s biosphere. Furthermore,optimisation of proteorhodopsin synthesis in vitro and from E. coli shows that yieldssufficient for structural studies by NMR and X-ray crystallography are achievable.Over-production of functional transhydrogenase with an altered sub-unit composition,where the β-subunit is split into two polypeptides, is presented. This construct wasutilised to confirm that transmembrane helix nine is adjacent to helices 13 and 14.Thus, it provides support for the suggestion that these three helices, presumablytogether with helix ten, form the proton translocation channel.An X-ray structure of intrinsic AcrB, the main cause of multi-drug resistance in gramnegativebacteria, is determined. Two novel structural features are observed: apreviously unsuspected transmembrane helix and a specific rotation of the periplasmicporter domain of AcrB. Using mass spectrometry the new helix is assigned asoriginating from a new subunit, the single transmembrane protein YajC. However,growth studies could not conclusively determine the functional role of YajC whenassociated with AcrB. Nevertheless, the observed twist might explain how TolC isopened to allow drug export from the cell, since this motion is consistent with thehypothesised "twists to open" mechanism for TolC activation

Generation and exploitation of proton motive force: Biochemical and structural analysis of three bacterial integral membrane proteins.

Proton motive force, a necessity for all living cells, is generated and exploited by asignificant number of membrane-bound enzymes and transporters. The focus of thisthesis was to understand the physiological relevance, structure and mechanism of threesuch bacterial enzymes: proteorhodopsin; transhydrogenase; and AcrB.Proteorhodopsin, common in surface living bacteria, is demonstrated to exist also inphylum Bacteriodetes, the third most abundant class of ocean bacteria. Evidence is alsopresented that its presence stimulates growth of one such bacterium in the presence oflight. This finding indicates that proteorhodopsin associated growth enhancementcould contribute to the primary production in earth’s biosphere. Furthermore,optimisation of proteorhodopsin synthesis in vitro and from E. coli shows that yieldssufficient for structural studies by NMR and X-ray crystallography are achievable.Over-production of functional transhydrogenase with an altered sub-unit composition,where the β-subunit is split into two polypeptides, is presented. This construct wasutilised to confirm that transmembrane helix nine is adjacent to helices 13 and 14.Thus, it provides support for the suggestion that these three helices, presumablytogether with helix ten, form the proton translocation channel.An X-ray structure of intrinsic AcrB, the main cause of multi-drug resistance in gramnegativebacteria, is determined. Two novel structural features are observed: apreviously unsuspected transmembrane helix and a specific rotation of the periplasmicporter domain of AcrB. Using mass spectrometry the new helix is assigned asoriginating from a new subunit, the single transmembrane protein YajC. However,growth studies could not conclusively determine the functional role of YajC whenassociated with AcrB. Nevertheless, the observed twist might explain how TolC isopened to allow drug export from the cell, since this motion is consistent with thehypothesised "twists to open" mechanism for TolC activation

Structural models of the human copper P-type ATPases ATP7A and ATP7B

The human copper exporters ATP7A and ATP7B contain domains common to all P-type ATPases as well as class-specific features such as six sequential heavy-metal binding domains (HMBD1-HMBD6) and a type-specific constellation of transmembrane helices. Despite the medical significance of ATP7A and ATP7B related to Menkes and Wilson diseases, respectively, structural information has only been available for isolated, soluble domains. Here we present homology models based on the existing structures of soluble domains and the recently determined structure of the homologous LpCopA from the bacterium Legionella pneumophila. The models and sequence analyses show that the domains and residues involved in the catalytic phosphorylation events and copper transfer are highly conserved. In addition, there are only minor differences in the core structures of the two human proteins and the bacterial template, allowing protein-specific properties to be addressed. Furthermore, the mapping of known disease-causing missense mutations indicates that among the heavy-metal binding domains, HMBD5 and HMBD6 are the most crucial for function, thus mimicking the single or dual HMBDs found in most copper-specific P-type ATPases. We propose a structural arrangement of the HMBDs and how they may interact with the core of the proteins to achieve autoinhibition

Crystallization and preliminary structural analysis of the Listeria monocytogenes Ca2+-ATPase LMCA1

Ca(2+)-ATPases are ATP-driven membrane pumps that are responsible for the transport of Ca(2+) ions across the membrane. The Listeria monocytogenes Ca(2+)-ATPase LMCA1 has been crystallized in the Ca(2+)-free state stabilized by AlF(4) (−), representing an occluded E2–P(i)-like state. The crystals belonged to space group P2(1)2(1)2 and a complete data set extending to 4.3 Å resolution was collected. A molecular-replacement solution was obtained, revealing type I packing of the molecules in the crystal. Unbiased electron-density features were observed for AlF(4) (−) and for shifts of the helices, which were indicative of a reliable structure determination

Isolation and Crystallization of the D156C Form of Optogenetic ChR2

Channelrhodopsins (ChRs) are light-gated ion channels that are receiving increasing attention as optogenetic tools. Despite extensive efforts to gain understanding of how these channels function, the molecular events linking light absorption of the retinal cofactor to channel opening remain elusive. While dark-state structures of ChR2 or chimeric proteins have demonstrated the architecture of non-conducting states, there is a need for open-and desensitized-state structures to uncover the mechanistic principles underlying channel activity. To facilitate comprehensive structural studies of ChR2 in non-closed states, we report a production and purification procedure of the D156C form of ChR2, which displays prolonged channel opening compared to the wild type. We demonstrate considerable yields (0.45 mg/g fermenter cell culture) of recombinantly produced protein using S. cerevisiae, which is purified to high homogeneity both as opsin (retinal-free) and as functional ChR2 with added retinal. We also develop conditions that enable the growth of ChR2 crystals that scatter X-rays to 6 Å, and identify a molecular replacement solution that suggests that the packing is different from published structures. Consequently, our cost-effective production and purification pipeline opens the way for downstream structural studies of different ChR2 states, which may provide a foundation for further adaptation of this protein for optogenetic applications

Crystal Structure of Na+, K+-ATPase in the Na+-Bound State

The Na(+), K(+)-adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na(+) and K(+) across the plasma membrane--a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K(+)-bound or ouabain-blocked forms. We present the crystal structure of a Na(+)-bound Na(+), K(+)-ATPase as determined at 4.3 Å resolution. Compared with the K(+)-bound form, large conformational changes are observed in the α subunit whereas the β and γ subunit structures are maintained. The locations of the three Na(+) sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na(+) from IIIb through IIIa, followed by exchange of Na(+) for K(+) at sites I and II, is suggested