| 1. |
- Bill, Roslyn M., et al.
(author)
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Overcoming barriers to membrane protein structure determination.
- 2011
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In: Nature biotechnology. - : Springer Science and Business Media LLC. - 1546-1696 .- 1087-0156. ; 29:4, s. 335-40
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Journal article (peer-reviewed)abstract
- After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.
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| 2. |
- Drew, David, et al.
(author)
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GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.
- 2008
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In: Nat Protoc. - 1750-2799. ; 3:5, s. 784-98
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Journal article (peer-reviewed)abstract
- It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 micro GFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.
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| 3. |
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| 4. |
- Lee, Chiara, et al.
(author)
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A two-domain elevator mechanism for sodium/proton antiport
- 2013
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 501:7468, s. 573-577
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Journal article (peer-reviewed)abstract
- Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets(2). The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli(1,3), for which both electron microscopy and crystal structures are available(4-6). NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein(1,4). Likemany Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur(7). The only reported NhaA crystal structure so far is of the low pH inactivated form(4). Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 angstrom resolution, solved from crystals grown at pH7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding(1,8,9) directly, a role supported hereby molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20 degrees against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second(3), Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.
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| 5. |
- Meier, Pascal F., 1987-
(author)
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Elucidating the molecular basis of Na+/H+ exchange
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Solute carrier (SLC) transporters are membrane transport proteins, which catalyse the movement of nutrients, ions, and drugs across cell membranes. Here, I will present our contribution to understanding the mechanism of the sodium/proton exchangers (NHE), belonging to the SLC9 family of membrane transporters. NHEs exchange sodium ions for protons across biological membranes, which is a critical reaction for the fine-tuning of cytoplasmic and organelle pH, sodium levels and volume homeostasis. Dysfunction of NHE members has been linked to a number of diseases and disorders, such as hypertension, heart failure, autism spectrum disorder, epilepsy and the susceptibility of long COVID. Protein structures are important for developing mechanistic models, but due to technical challenges only bacterial homologue structures of NHE proteins were previously available.Accumulating many years of effort, we were able to determine the first structure of a mammalian Na+/H+ exchanger, the endosomal isoform NHE9 by single-particle cryo-EM. The structure of NHE9 demonstrated that NHE proteins are architecturally most similar to bacterial homologues with 13-TM segments and likely operated by a similar elevator mechanism (I). Interestingly, native MS and thermal-shift assays indicted that the NHE9 homodimer is stabilized by the binding of a rare lipid only found in late endosomes, which implies the cell may use this lipid as means to switch-on NHE9 activity once it reaches its correct functional localization. We further provided evidence that the large cytoplasmic tail in NHE proteins likely acts in an auto-inhibitory manner. It is only removed upon the binding of extrinsic proteins (II). Indeed, the first structure of a potassium specific K+/H+ exchanger KefC reveals how its cytoplasmic tail restricts movement of the ion-transporting domain to directly inhibit transport. The structure of KefC is also the first ion-bound state seen for this family and, unlike to the modeled Na+/H+ exchanger sites with a hydrated Na+ ion, coordinates K+ as a dehydrated ion (IV). Lastly, we determining the structure of a bacterial Na+/H+ exchanger NhaA to high-resolution at an active pH of 6.5. With this structure we demonstrated how a cytoplasmic “pH gate” controlled by the pH activated NhaA (III).
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| 6. |
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| 7. |
- Skaar, Karin, 1981-
(author)
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Structural and biochemical studies of phage P2 DNA-binding proteins and human tetraspanins
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Biochemical studies of proteins are crucial for a more detailed view of the world around us. The focus of biochemical studies can vary, from a complex mammalian system to a more simple viral entity, but the same methods and principles apply. In biochemistry one rely on both in vitro and in vivo analyses to understand biological processes. Protein crystallography has since the late 1950s been an additional important tool. By visualizing the structures of molecules involved in a biological process one can truly comprehend the molecular mechanisms of an organism or cell at the chemical level. This thesis includes structural biochemical work in combination with mutational and functional studies of proteins from both human and virus.Human tetraspanins are integral membrane proteins grouped by their conserved structural features. Many of them have been shown to regulate cell migration, fusion, and signalling in the cell by functioning as organizers of multi-molecular membrane complexes. Several tetraspanins are also implicated in different forms of human cancers. How tetraspanins perform their function is still not known at the molecular level and today very little structural data exist on complete tetraspanin proteins. Structural biochemical studies require mg quantities of purified protein, something that is not easily obtained for membrane proteins. This thesis includes a family-wide approach to achieve full-length tetraspanins for biochemical studies. To facilitate this process a GFP-based optimization scheme for production and purification of membrane proteins in E. coli and S. cerevisiae has been applied. By utilizing this approach, we identified 8 human tetraspanins that can be produced and isolated from either E. coli or S. cerevisiae, and in one case using either system.The temperate bacteriophage P2 is a virus, which can enter both the lytic and the lysogenic cycle upon infection of its host. The outcome of the infection is regulated by and dependent on several proteins encoded by the viral genome. The immunity repressor P2 and the Cox repressor direct the phage into either cycle. Integration and excision of the virus DNA requires the enzyme P2 integrase. The work in this thesis presents high-resolution crystal structures of these key proteins from the regulation of lysogeny in bacteriophage P2. By using a crystallographic approach in combination with mutational studies, key characteristics of these three proteins are presented.
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