B. Göran Karlsson

Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli

The structural gene for the blue copper protein azurin from Pseudomonas aeruginosa has been subcloned in different expression plasmid vectors. The highest yield of expression was obtained when the gene with its native ribosome‐binding site was placed downstream of the lac promoter in plasmid pUC18. The protein is exported to the periplasmic space in Escherichia coli and the amount corresponds to 27% of the total protein content in the periplasmic space. The preprotein is cleaved correctly according to N‐terminal sequencing of the purified protein. Azurin has been purified in large amounts and is spectroscopically indistinguishable from the protein purified from P. aeruginosa.

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).

Rack-induced bonding in blue copper proteins: spectroscopic properties and reduction potential of the azurin mutant Met-121 Leu

Site‐directed mutagenesis has been used to prepare azurin in which the methionine‐121 residue has been replaced by leucine. The oxidized mutant protein displays the strong blue color and characteristic EPR signal of a type 1 Cu(II) ion, showing that methionine is not an obligatory component of a blue copper site. The optical absorption maximum is shifted 5 nm towards longer wavelength and the extinction coefficient increased by about 10% compared to the wild‐type protein. In addition, there are small changes in the EPR parameters, in particular the copper hyperfine splitting. The reduction potential is increased by 70 mV. The results show that a small change in primary structure without any alteration in the three strong ligands can perturb the Cu(II) site and shift the reduction potential, in accord with the concept of rack‐induced bonding in blue copper proteins.

Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties

Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP(+) to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains. Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively. Domain II spans the membrane and constitutes at least partly the proton translocating pathway. Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented. The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP(+) by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site.

Reduction potentials of blue and purple copper proteins in their unfolded states: a closer look at rack-induced coordination

Abstract Cyclic voltammetry has been used to determine the reduction potentials of blue (Pseudomonas aeruginosa azurin) and purple (Thermus thermophilus CuA domain) copper proteins unfolded by guanidine hydrochloride. These Cu(II/I) potentials [456 (azurin); 453 (CuA) mV vs., NHE] are higher than those of the folded proteins. The downshift of the potential in the folded state can be accounted for by assuming that rack-induced axial coordination stabilizes Cu(II) relative to Cu(I) in a protein-encapsulated active site.

Rational improvement of cell-free protein synthesis.

Experimental design principles were applied on cell-free protein synthesis to optimize performance with regard to the expression yield and the incorporation efficiency of amino acid precursors. A versatile screening platform based on batch-mode cell-free expression and central composite design was used. The performance of different extracts (S12 and S30), the concentration dependence of key components and the effect of different additives were investigated. We find that the initial expression yield can be enhanced twofold to threefold in this manner. The improved conditions comprise a modified S12 extract, optimized concentrations of creatine phosphate and key amino acids, as well as introduction of ketoacid additives. Our results show that current cell-free expression technology is far from optimal and that higher yields and increased utilization of the provided precursors are attainable with further optimization.

Highly Efficient NMR Assignment of Intrinsically Disordered Proteins: Application to B- and T Cell Receptor Domains

We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution.

NMR detection of multiple transitions to low-populated states in azurin

Transitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone 15N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8°C). Two exchange processes with different rate constants could be discriminated despite populations of the excited states below 1% and spatial neighborhood of the two processes. The group of 15N nuclei involved in the faster process exhibits at 44.8°C a forward rate constant of 11.7±2.4 s−1 and a population of the exited state of 0.39±0.07%. They surround the aromatic ring of histidine 35 whose protonation state is coupled to the flipping of a neighboring peptide plane. For the slower process, the forward rate constant and population of the exited state at 44.8°C are 4.1±0.1 s−1 and 0.45±0.02%, respectively. The residues involved cluster nearby the copper ion, which is separated from the protonation site of histidine 35 by about 8 Å, indicating conformational rearrangements involving the copper coordinating loops. The dependence of the equilibrium constant on the temperature is consistent with an enthalpy‐dominated transition around the copper, but an entropy‐controlled transition near histidine 35. The detection by nuclear magnetic resonance of millisecond to second conformational transitions near the copper ion suggests a low energy‐cost rearrangement of the copper‐binding site that may be necessary for efficient electron transfer.

Electronic absorption spectra of M(II)(Met121X) azurins (MCo, Ni, Cu; XLeu, Gly, Asp, Glu): charge-transfer energies and reduction potentials

Abstract Electronic absorption spectra of the Co(II) and Ni(II) derivatives of Met121X (XLeu, Gly, Asp, Glu) azurin mutants have been measured. Coordination of carboxylate to the metal ion is indicated by LF and LMCT band shifts in the Met121Glu proteins. The relatively low reduction potentials of the Cu(II)(Met121X) (XAsp, Glu) azurins accord with the LMCT energies of the corresponding Co(II) derivatives.

Expression screening of membrane proteins with cell-free protein synthesis

Detailed biophysical studies of integral membrane proteins are often hampered by sample preparation difficulties. Membrane proteins are typically difficult to express in sufficient amounts to enable the use of demanding techniques such as nuclear magnetic resonance and X-ray crystallography for structural biology. Here, we show that an inexpensive batch-based cell-free expression system can be a viable alternative for production of a wide range of different membrane proteins, both of prokaryotic and eukaryotic origin. Out of 38 tested protein constructs, 37 express at levels suitable for structural biology, i.e. enough to produce several milligrams of protein routinely and without excessive costs. This success rate was not anticipated and is even more impressive considering that more than half of the expressed proteins where of mammalian origin. A detergent screen identified Brij-58 as the, in general, most successful choice for co-translational solubilization of the expressed proteins.