Expression of nuclear hormone receptors in Escherichia coli.

Nuclear hormone receptors are ligand-activated transcription factors. The study of nuclear hormone receptor--ligand interactions requires expression of soluble receptor, but the production of functional soluble receptors in E. coli has generally proved difficult. Recent studies on coexpression of nuclear hormone receptors have yielded active soluble proteins, which were not readily generated when expressed individually.

Effects of intracerebroventricular infusion of leptin in obese Zucker rats.

The obese Zucker rat (OZR) exhibits a missense mutation in the cDNA for the leptin receptor, producing a single amino acid substitution in the extracellular domain of the receptor. A mutation in the leptin receptor gene of the db/db mouse prevents the synthesis of the long splice variant of the receptor. The possibility that the OZR, like the db/db mouse, is refractory to the actions of murine leptin was tested by infusing the protein intracerebroventricularly via a minipump for 7 days. Lean Zucker rats (LZR) infused with leptin acted as positive controls, and other groups of OZR and LZR were infused with vehicle. In LZR, leptin reduced bodyweight and food intake and increased brown adipose tissue (BAT) temperature. Plasma corticosterone increased (61%) in these rats, and plasma triglycerides fell (78%). Leptin treatment improved tolerance to an oral glucose load (16% reduction in the area under the blood glucose curve) while lowering plasma insulin. In OZR, the actions of leptin were blunted. Food intake was slightly, but not significantly, reduced. Although there was a reduction in the rate of increase in body mass, the effect of leptin was about half that seen in LZR. BAT temperature and glucose tolerance were unchanged. In contrast to the elevated plasma corticosterone seen in LZR, leptin reduced the level of this hormone (27%) in OZR. In OZR and LZR treated with leptin, the plasma leptin levels were increased 24-fold and 47-fold, respectively. The results suggest that leptin retains some efficacy in OZR, although these rats are less responsive than LZR.

NMR analysis of main-chain conformational preferences in an unfolded fibronectin-binding protein.

A 130-residue fragment of the Staphylococcus aureus fibronectin-binding protein has been found to exist in a highly unfolded conformation at neutral pH. Measurement of experimental NMR 3JHNalpha coupling constants provides evidence for individual residues having distinct main-chain conformational preferences that are dependent both on the amino acid concerned and on neighbouring residues in the sequence. Analysis shows that these variations in the populations of individual residues can be explained in detail in terms of statistical distributions of conformational states derived from the protein data base. In particular, when the preceding residue has a beta-branched or aromatic side-chain, a significant increase occurs in the population of the less sterically restricted b region of phi,psi space. The results indicate that the local structure of the fibronectin binding protein in solution, under conditions where it displays full activity, approximates very closely to a statistical random coil structure. This may be an important feature in the biological role of this and other polypeptides involved in protein-protein interactions.

Folding and conformational studies on SCR1-3 domains of human complement receptor 1.

Short consensus repeats SCR3 and SCR1-3 are soluble recombinant proteins, consisting of the third and first three N-terminal domains of complement receptor 1, respectively, which retain some anti-complement activity. The conformational stabilities and folding/unfolding of SCR3 and SCR1-3 have been studied using circular dichroism and equilibrium and pre-equilibrium fluorescence spectroscopy. Denaturation by guanidinium hydrochloride (GdnHCl) is rapid and completely reversible. Reduction of disulphide bridges in the folded proteins by beta-mercaptoethanol leads to an increase in fluorescence intensity. The fluorescence intensity of the folded proteins is approximately 7.5% of that of the respective unfolded proteins. The data can be approximated to a two-state transition between native and denatured forms of the proteins. SCR3 has a conformational stability in water of 12-13 kJ/mol whereas that of SCR1-3 is 19.5-19.9 kJ/mol depending upon the technique utilized. The heat capacity change associated with the unfolding of SCR1-3 was obtained by a series of GdnHCl unfolding experiments over a range of temperatures and was found to be 6.6 kJ/K.mol or 33.8 J/K.mol(residue). The refolding process of SCR3 was found to be simple, described by a single exponential equation, whereas that of SCR1-3 was found to be complex and could be fitted to a double exponential equation indicating the presence of folding intermediates.

Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1.

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system in Escherichia coli was used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP-Sepharose. The oligomer was folded by one-step dilution in 20 mM ethanolamine/1 mM EDTA supplemented with 1 mM GSH/0.5 mM GSSG. The folded material was processed to a concentrated (> 20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1-3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 x 110 A. SCR(1-3) has an unusual CD spectrum exhibiting a broad maximum at 220-230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.

Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis.

Barnase, the ribonuclease from Bacillus amyloliquefaciens, has been cloned and expressed in Escherichia coli [Hartley, R. W. (1988) J. Mol. Biol. 202, 913-915], thus enabling the overproduction and site-directed mutagenesis of one of the smallest enzymes (Mr equals 12,382). As barnase is also composed of just a single polypeptide chain with no disulfide bridges and has a reversible folding transition, it affords a fine system for studying protein folding and design. We show here that the recombinant enzyme has properties identical with those of the authentic enzyme, characterize the basic kinetics and specificity of the enzyme, and, using site-directed mutagenesis, identify key residues involved in catalysis to provide evidence that supports the classic ribonuclease mechanism. The wild-type enzyme catalyzes the hydrolysis of dinucleotides of structure GpN. There is a prime requirement for G and a preference for A greater than G greater than C greater than U for N. The pH-activity curve for the transesterification step of dinucleotides is bell shaped with an optimum for kcat/KM and kcat at about pH 5. The enzyme is far more active toward long RNA molecules, and the pH optimum for kcat is at 8.5. The activity of barnase toward dinucleotide substrates is about 0.5% of that of the highly homologous T1 nuclease at pH 5.9, but barnase is twice as active as T1 toward RNA at pH 8.5. There must be important subsite interactions that contribute to catalysis in barnase in addition to those immediately on either side of the scissile bond.(ABSTRACT TRUNCATED AT 250 WORDS)