Nicklas Bonander

The effect of the metal ion on the folding energetics of azurin: a comparison of the native, zinc and apoprotein.

The unfolding by guanidine hydrochloride (GuHCl) and the refolding on dilution of zinc and apoazurin have been monitored by far-UV circular dichroism (CD). With the native protein, the unfolding was followed by CD and optical absorption in the visible spectral region. With the zinc protein, the reversible unfolding has also been followed by tryptophan fluorescence and NMR. The zinc and Cu2+ metal ions remain associated with the protein in the unfolded state. When the unfolding of the native protein is followed by CD, the initial, reversible transition due to unfolding is followed by a slow change associated with the reduction of Cu2+ by the thiol group of the ligand Cys112. The unfolding of apoazurin displays two CD transitions, which evidence suggests represent different folding domains, the least stable one including the metal-binding site and the other one the rest of the beta-sheet structure. Both occur at a lower GuHCl concentration than the unfolding of the native protein. The CD titrations also demonstrate that zinc azurin has a lower stability than the copper protein. Unfolding of zinc azurin followed by tryptophan fluorescence occurs at a much lower GuHCl concentration than the CD changes, and NMR spectra show that there is no loss of secondary and tertiary structure at this concentration, whereas the CD-detected loss of secondary structure correlates with the NMR changes. Thus, the fluorescence change is ascribed to a small local perturbation of the structure around the single tryptophan residue. The differences in stability of the three forms of azurin are discussed in terms of the rack mechanism. A bound metal ion stabilizes the native fold, and this stabilization is larger for Cu(II) than for Zn(II), reflecting the higher affinity of the protein for Cu(II).

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.

The effect of redox state on the folding free energy of azurin

Abstract The unfolding of oxidized and reduced azurin by guanidine hydrochloride has been monitored by circular dichroism. Dilution experiments showed the unfolding to be reversible, and the equilibrium data have been interpreted in terms of a two-state model. The protein is stabilized by the strong metal binding in the native state, so that the folding free energy is as high as –52.2 kJ mol–1 for the oxidized protein. The reduced protein is less stable, with a folding free energy of –40.0 kJ mol–1. A thermodynamic cycle shows, as a consequence, that unfolded azurin has a reduction potential 0.13 V above that of the folded protein. This is explained by the bipyramidal site in the native fold stabilizing Cu(II) by a rack mechanism, with the same geometry being maintained in the Cu(I) form. In the unfolded protein, on the other hand, the coordination geometries are expected to differ for the two oxidation states, Cu(I) being stabilized by the cysteine thiol group in a linear or trigonal symmetry, whereas Cu(II) prefers oxygen ligands in a tetragonal geometry.

Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump

Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.

Crystal structure of the disulfide bond-deficient azurin mutant C3A/C26A: how important is the S-S bond for folding and stability?

Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 A resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 M GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol-1, a destabilization of approximately 20 kJ.mol-1 compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.

The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal structure determination of the Co(II) derivative and Co-EPR spectroscopy.

The crystal structure of cobalt‐substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 Å resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild‐type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild‐type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,Sδ atom is increased to 3.3–3.5 Å and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1–2.4 Å. The X‐band EPR spectrum of the high‐spin Co(II) in azurin is well resolved (apparent g values gx′ = 5.23; gy′ = 3.83; gz′ = 1.995, and hyperfine splittings Ax′ = 31; Ay′ = 20–30; Az′ = 53 G) and indicates that the ligand field is close to axial. Proteins 27:385–394, 1997.

Improved sugar co-utilisation by encapsulation of a recombinant Saccharomyces cerevisiae strain in alginate-chitosan capsules

BackgroundTwo major hurdles for successful production of second-generation bioethanol are the presence of inhibitory compounds in lignocellulosic media, and the fact that Saccharomyces cerevisiae cannot naturally utilise pentoses. There are recombinant yeast strains that address both of these issues, but co-utilisation of glucose and xylose is still an issue that needs to be resolved. A non-recombinant way to increase yeast tolerance to hydrolysates is by encapsulation of the yeast. This can be explained by concentration gradients occuring in the cell pellet inside the capsule. In the current study, we hypothesised that encapsulation might also lead to improved simultaneous utilisation of hexoses and pentoses because of such sugar concentration gradients.ResultsIn silico simulations of encapsulated yeast showed that the presence of concentration gradients of inhibitors can explain the improved inhibitor tolerance of encapsulated yeast. Simulations also showed pronounced concentration gradients of sugars, which resulted in simultaneous xylose and glucose consumption and a steady state xylose consumption rate up to 220-fold higher than that found in suspension culture. To validate the results experimentally, a xylose-utilising S. cerevisiae strain, CEN.PK XXX, was constructed and encapsulated in semi-permeable alginate-chitosan liquid core gel capsules. In defined media, encapsulation not only increased the tolerance of the yeast to inhibitors, but also promoted simultaneous utilisation of glucose and xylose. Encapsulation of the yeast resulted in consumption of at least 50% more xylose compared with suspended cells over 96-hour fermentations in medium containing both sugars. The higher consumption of xylose led to final ethanol titres that were approximately 15% higher. In an inhibitory dilute acid spruce hydrolysate, freely suspended yeast cells consumed the sugars in a sequential manner after a long lag phase, whereas no lag phase was observed for the encapsulated yeast, and glucose, mannose, galactose and xylose were utilised in parallel from the beginning of the cultivation.ConclusionsEncapsulation of xylose-fermenting S. cerevisiae leads to improved simultaneous and efficient utilisation of several sugars, which are utilised sequentially by suspended cells. The greatest improvement is obtained in inhibitory media. These findings show that encapsulation is a promising option for production of second-generation bioethanol.

Optimising Yeast as a Host for Recombinant Protein Production (Review)

Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the proteins native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.

Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Crystal structure of the double azurin mutant Cys3Ser/Ser100Pro from Pseudomonas aeruginosa at 1.8 Å resolution: its folding–unfolding energy and unfolding kinetics

Azurin is a cupredoxin, which functions as an electron carrier. Its fold is dominated by a beta-sheet structure. In the present study, azurin serves as a model system to investigate the importance of a conserved disulphide bond for protein stability and folding/unfolding. For this purpose, we have examined two azurin mutants, the single mutant Cys3Ser, which disrupts azurins conserved disulphide bond, and the double mutant Cys3Ser/Ser100Pro, which contains an additional mutation at a site distant from the conserved disulphide. The crystal structure of the azurin double mutant has been determined to 1.8 A resolution(2), with a crystallographic R-factor of 17.5% (R(free)=20.8%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. Also, the rates of folding and unfolding as determined by CD and fluorescence spectroscopy are almost unchanged. The main difference to wild-type azurin is a destabilisation by approximately 20 kJ x mol(-1), constituting half the total folding energy of the wild-type protein. Thus, the disulphide bond constitutes a vital component in giving azurin its stable fold.