Kristina Hedfalk

Structural mechanism of plant aquaporin gating

Plants counteract fluctuations in water supply by regulating all aquaporins in the cell plasma membrane. Channel closure results either from the dephosphorylation of two conserved serine residues under conditions of drought stress, or from the protonation of a conserved histidine residue following a drop in cytoplasmic pH due to anoxia during flooding. Here we report the X-ray structure of the spinach plasma membrane aquaporin SoPIP2;1 in its closed conformation at 2.1u2009Å resolution and in its open conformation at 3.9u2009Å resolution, and molecular dynamics simulations of the initial events governing gating. In the closed conformation loop D caps the channel from the cytoplasm and thereby occludes the pore. In the open conformation loop D is displaced up to 16u2009Å and this movement opens a hydrophobic gate blocking the channel entrance from the cytoplasm. These results reveal a molecular gating mechanism which appears conserved throughout all plant plasma membrane aquaporins.

Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris

The aquaporin PM28A is one of the major integral proteins in spinach leaf plasma membranes. Phosphorylation/dephosphorylation of Ser274 at the C‐terminus and of Ser115 in the first cytoplasmic loop has been shown to regulate the water channel activity of PM28A when expressed in Xenopus oocytes. To understand the mechanisms of the phosphorylation‐mediated gating of the channel the structure of PM28A is required. In a first step we have used the methylotrophic yeast Pichia pastoris for expression of the pm28a gene. The expressed protein has a molecular mass of 32462 Da as determined by matrix‐assisted laser desorption ionization‐mass spectrometry, forms tetramers as revealed by electron microscopy and is functionally active when reconstituted in proteoliposomes. PM28A was efficiently solubilized from urea‐ and alkali‐stripped Pichia membranes by octyl‐β‐D‐thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.

Eukaryotic expression: developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high‐throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus‐infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein‐production pipelines are reported. Strategies for co‐expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Design of improved membrane protein production experiments: Quantitation of the host response

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial‐and‐error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high‐performance bioreactors under tightly‐defined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly‐controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology.

Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations

ABSTRACT The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Δ strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a non-ethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Δ strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain.

Production, characterization and crystallization of the Plasmodium falciparum aquaporin.

The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A+T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18mg/L) and initial attempts at crystallization of the protein yielded several different forms.

Overexpression and Purification of the Glycerol Transport Facilitators, Fps1p and GlpF, in Saccharomyces Cerevisiae and Escherichia Coli

Fpslp is an osmoregulated glycerol transport facilitator, located in the Saccharomyces cerevisiae plasma membrane, where it is mainly used for efflux of glycerol in the adaptation of yeast cells to lower external osmolarity (Luyten et al., 1995). Fpslp is an unusual member of the MIP (Major Intrinsic Protein)-family. The protein is much larger than most of the other proteins in the family, due to long hydrophilic extensions at both termini. The N-terminus (~250 aa) is involved in the regulation of the channel (Tamas et al., 1999), but the function of the C-terminus (~150 aa) is not yet known. Fpslp is also one of the few members that differs in the well-conserved NPA-motifs in the two putative channel forming loops B and E; serine instead of alanine in Loop B and leucine instead of proline in Loop E.

Design of improved membrane protein production experiments in yeast: quantitation of the host response

Address: 1Department of Cell and Molecular Biology/Microbiology, Göteborg University, Box 462, 405 30 Göteborg, Sweden, 2Department of Chemistry and Bioscience/Molecular Biotechnology, Chalmers University of Technology, Box 462, 405 30 Göteborg, Sweden, 3Department of Mathematics, Chalmers University of Technology, 41296 Göteborg, Sweden, 4The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA and 5School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK * Corresponding author