123 research outputs found
Initiating Heavy-atom Based Phasing by Multi-Dimensional Molecular Replacement
To obtain an electron-density map from a macromolecular crystal the
phase-problem needs to be solved, which often involves the use of heavy-atom
derivative crystals and concomitantly the determination of the heavy atom
substructure. This is customarily done by direct methods or Patterson-based
approaches, which however may fail when only poorly diffracting derivative
crystals are available, as often the case for e.g. membrane proteins. Here we
present an approach for heavy atom site identification based on a Molecular
Replacement Parameter Matrix (MRPM) search. It involves an n-dimensional search
to test a wide spectrum of molecular replacement parameters, such as clusters
of different conformations. The result is scored by the ability to identify
heavy-atom positions, from anomalous difference Fourier maps, that allow
meaningful phases to be determined. The strategy was successfully applied in
the determination of a membrane protein structure, the CopA Cu+-ATPase, when
other methods had failed to resolve the heavy atom substructure. MRPM is
particularly suited for proteins undergoing large conformational changes where
multiple search models should be generated, and it enables the identification
of weak but correct molecular replacement solutions with maximum contrast to
prime experimental phasing efforts.Comment: 19 pages total, main paper: 6 pages (2 figures), supplementary
material: 13 pages (2 figures, 9 tabels
A sulfur-based transport pathway in Cu^+-ATPases
Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. P_(IB)âtype Cu^+âATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu^+ across cellular membranes. Crystal structures of a copperâfree Cu^+âATPase are available, but the mechanism of Cu^+ recognition, binding, and translocation remains elusive. Through Xâray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solidâsupported membranes using wildâtype and mutant forms of the Legionella pneumophila Cu^+âATPase (LpCopA), we identify a sulfurâlined metal transport pathway. Structural analysis indicates that Cu^+ is bound at a highâaffinity transmembraneâbinding site in a trigonalâplanar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the highâaffinity site and subsequent release through the exit pathway
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
Assessing water permeability of aquaporins in a proteoliposome-based stopped-flow setup
Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux. The consequent effects on proteoliposome size can be tracked using the fluorescence of an internalized fluorophore. This enables controlled characterization of water flux by AQPs. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020). [Abstract copyright: Š 2022 The Authors.
High-yield overproduction and purification of human aquaporins from Pichia pastoris
Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1â5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014)
Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema
Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies
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