Evaluation of refolding conditions for a recombinant human interleukin-3 variant (daniplestim).

The refolding of daniplestim, a human interleukin-3 variant (SC-55494) from Escherichia coli inclusion bodies, was optimized using a reversed-phase HPLC method developed to permit quantification of the reduced and oxidized forms of daniplestim. The presence of cysteine or dithiothreitol accelerated refolding of daniplestim from E. coli inclusion body slurries dissolved in urea or guanidine solutions and was complete in 4-6 h. Regardless of the dissolution and refolding protocol used to renature daniplestim, equivalently bioactive protein was produced. Under refolding conditions, no covalent modification of daniplestim by cysteine or cyanate was observed. The folding process was characterized further by following the unfolding of purified daniplestim by far-UV CD and fluorescence spectroscopies under both oxidizing and reducing conditions at pH values between 7 and 11. Formation of the single disulphide bond had a large stabilizing effect on daniplestim structure ( approximately 4-5 kCal at pH 9.5). This thermodynamic stabilization drove the refolding process towards the native form, even under conditions where the reduced protein was largely unfolded. From these data, scaleable refolding conditions for daniplestim were established.

Energetic components of the allosteric machinery in hemoglobin measured by hydrogen exchange.

A hydrogen exchange (HX) functional labeling method was used to study allosterically active segments in human hemoglobin (Hb) at the alpha-chain N terminus and the beta-chain C terminus. Allosterically important interactions that contact these segments were removed one or more at a time by mutation (Hbs Cowtown, Bunbury, Barcelona, Kariya), proteolysis (desArg141alpha, desHis146beta), chemical modification (N-ethylsuccinimidyl-Cys93beta), and the withdrawal of extrinsic effectors (phosphate groups, chloride). The effects of each modification on HX rate at the local and the remote position were measured in the deoxy Hb T-state and translated into change in structural free energy at each position.The removal of individual salt links destabilizes local structure by 0.4 to 0.75 kcal/mol (pH 7.4, 0 degreesC, 0.35 M ionic strength) and often produces cross-subunit effects while hemoglobin remains in the T-state. In doubly modified hemoglobins, different changes that break the same links produce identical destabilization, changes that are structurally independent show energetic additivity, and changes that intersect show energetic overlap. For the overall T-state to R-state transition and for some but not all modifications within the T-state, the summed loss in stabilization free energy measured at the two chain termini matches the total loss in allosteric free energy measured by global methods. These observations illustrate the importance of evaluating the detailed energetics and the modes of energy transfer that define the allosteric machinery.

Site-directed mutagenesis of recombinant bovine placental lactogen at lysine-73 leads to selective attenuation of its somatogenic activity.

Bovine placental lactogen (bPL) is capable of binding and transducing biological activity via somatogenic and lactogenic receptors. To modify this capability, three analogs, bPL(K73D), bPL(K73F) and bPL(K73A), mutated at position 73, and corresponding to R64 in human GH (hGH), were produced in Escherichia coli. Circular dichroic spectrum analyses indicated proper refolding in all cases. Biological activity of these analogs was tested in vitro. In a lactogenic-receptor-mediated Nb2 rat lymphoma cell bioassay, bPL and its analogs acted similarly. In another lactogenic bioassay that measures beta-casein synthesis by HC-11 mouse mammary-gland cells, the analogs were 30-40% as potent as bPL. In contrast, somatogenic receptor-mediated bioactivity in FDC-P1 cells transfected with either rabbit (rb) or hGH receptor (R) was almost completely abolished in these analogs. In receptor binding assays, the effect was more conspicuous and the mutations affected not only somatogenic but also lactogenic binding. Binding to rat (r) and rabbit PRL receptor extracellular domains (ECDs) or membrane-embedded receptors was only slightly changed, except for bPL (K73D), which displayed very low affinity. In somatogenic binding assays to intact IM-9 human lymphocytes, hGHR-ECD or bovine liver membranes, bPL (K73D) did not bind at all, and bPL(K73F) or bPL(K73A) binding was drastically reduced. Binding experiments performed in real time using a BIAcore apparatus revealed that the decreased binding could be mainly attributed to increased k(off) rather than decreased k(on) values. The complex with hGHR-ECD revealed a 2:1 stoichiometry with bPL, bPL(K73F) and bPL(K73A), although the complex with these analogs was less stable than with bPL, whereas bPL(K73D) scarcely assembled a 1:1 complex. In contrast, bPL and the three analogs formed stable 1:2 complexes with rPRL-ECD. These results suggest that position 73 in bPL is more important for somatogenic than lactogenic properties and concurs with results from other groups, which have shown that R64, the analogous amino acid in hGH holds the same differential importance with respect to somatogenic binding.

Physical and kinetic characterization of recombinant human cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (MW) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre.g-1.cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% alpha-helix, 35% beta-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in 'half-reaction' experiments.

Selective modification of recombinant bovine placental lactogen by site-directed mutagenesis at its C terminus.

Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity. Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F). Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity. Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained. This selective modification most likely results from a steric hindrance induced by a bulky Phe-188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs. Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR. In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs. Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors.

Hydrogen exchange measurement of the free energy of structural and allosteric change in hemoglobin.

The inability to localize and measure the free energy of protein structure and structure change severely limits protein structure-function investigations. The local unfolding model for protein hydrogen exchange quantitatively related the free energy of local structural stability with the hydrogen exchange rate of concerted sets of structurally related protons. In tests with a number of modified hemoglobin forms, the loss in structural free energy obtained locally from hydrogen exchange results matches the loss in allosteric free energy measured globally by oxygen-binding and subunit dissociation experiments.

Porphyrin induced Z to B conversion of poly(dG-dC)2 in ethanol.

The water soluble porphyrins H2TMpyP-2, H2TMpyP-4, and CuTMpyP-4 are found to bind to Z-form poly(dG-dC)2 in 60% ethanol (v/v) and to facilitate the conversion of the polymer to the B form. Metalloporphyrins with axial ligands (MnTMpyP-4, ZnTMpyP-4) interact to some degree with the Z form, but do not lead to extensive conversion to the B form. The conversion of the Z form into the B form was determined by CD titration experiments, which were used to quantitate the fraction of poly(dG-dC)2 present in each conformation. Under all conditions each bound porphyrin molecule converts multiple base pairs from Z to B. The kinetics of porphyrin reactions with Z-poly(dG-dC)2 in 60% ethanol were measured using two different detection techniques. Stopped flow spectrophotometry was used to observe the time-dependent spectral changes associated with the porphyrins during the reaction. Time-dependent changes in the poly(dG-dC)2 conformation were observed directly using CD. The porphyrin absorbance changes under the conditions of these experiments have a much shorter half time (t1/2 approximately 0.1 to 2 sec) than the CD changes (t1/2 approximately 10 sec). Thus it could be determined that a complex with spectral characteristics similar to those of the porphyrin intercalated into B-form poly(dG-dC)2 is produced while the polymer is predominantly in the Z form.

Interactions of porphyrins with purified DNA and more highly organized structures.

Studies of the solution properties of gold(III)tetrakis(4-N-methylpyridyl) porphine and its DNA binding characteristics have been conducted utilizing uv/vis absorption spectroscopy, circular dichroism (CD), Mossbauer spectroscopy, and temperature-jump relaxation techniques. These studies indicate that over the concentration range considered this water soluble gold(III) porphyrin does not aggregate, binds axial ligands only weakly with a preference for soft Lewis bases, and is capable of intercalation into nucleic acids of appropriate base pair content. The interaction of this and several other porphyrins with the synthetic polynucleotide poly(dA-dC).poly(dT-dG) has been studied. Spectroscopic signatures for intercalation were found for those derivatives not having axial ligands. Intercalation into chromatin in vitro can also occur with those porphyrins and metalloporphyrins which do not have axial ligands. Finally, studies utilizing microinjection techniques indicate that once within the cell, tetrakis(4-N-methylpyridyl)porphine tends to localize in the nucleus.

A kinetic description of ligand binding to sperm whale myoglobin.

Nanosecond recombination time courses were measured by photolyzing O2, NO, CO, methyl, ethyl, n-propyl, n-butyl, and tert-butyl isocyanide complexes of sperm whale myoglobin with a 30-ns laser pulse at pH 7, 20 degrees C. Absorbance was measured both during and after the excitation pulse and as a function of laser light intensity. The results were analyzed quantitatively in terms of a three-step reaction scheme, MbX in equilibrium B in equilibrium C in equilibrium Mb + X, where Mb is myoglobin, B represents a geminate state in which the ligand is present in the distal pocket but not covalently bound to the iron atom, and C, a state in which the ligand is still embedded in the protein but further away from the heme group. The fitted rate parameters were required to be consistent with the observed overall quantum yield, Q, which had been measured independently using much longer (approximately 0.5 ms) xenon flash pulses. Three major conclusions were derived from these analyses. First, the overall quantum yield of the ligand complex is determined primarily by the competition between the rate of iron-ligand bond formation from the initial photoproduct, kB----MbX, and the rate of migration away from state B, kB----C. For example, kB----C approximately equal to 30-100 microseconds-1 for all three gaseous ligands, whereas both Q and kB----MbX vary over 3 orders of magnitude (i.e. NO, Q = 0.001, kB----MbX approximately equal to 16,000 microseconds-1; O2, Q = 0.1, kB----MbX approximately equal to 500 microseconds-1; CO, Q = 1.0, kB----MbX approximately equal to 2 microseconds-1). Second, for NO, O2, and the isonitriles, the rate-limiting step in the overall association reaction starting from ligand in solution is the formation of state B. The rate constant for this process varies from 2 X 10(7) M-1 s-1 for the gaseous ligands to 0.02-1.4 X 10(5) M-1 s-1 for the isonitriles. In contrast, the B to MbX transition is limiting for CO binding. Third, for all the ligands except CO, the overall rate of dissociation is limited significantly both by the rate of thermal bond disruption, kMbX----B, and the competition between geminate recombination and migration away from the distal pocket (i.e. kB----C/(kB----MbX + kB----C]. In the case of CO, the rate of bond disruption is equal to the observed dissociation rate constant.

Effects of solvent composition and viscosity on the rates of CO binding to heme proteins.

The rate of CO binding to myoglobin increases 4-fold, from 5 X 10(5) M-1 s-1 to 2 X 10(6) M-1 s-1, in going from 0 to 80% glycerol in phosphate buffer at pH 7, 20 degrees C. Under the same conditions, the rate of CO binding to protoheme decreases monotonically from about 1 X 10(8) M-1 s-1 to 2 X 10(7) M-1 s-1. The kinetic behavior of protoheme at neutral pH is that expected for a diffusion-controlled reaction. Increasing solvent viscosity causes a decrease in the observed second order rate constant. In contrast, the behavior of the myoglobin indicates quite clearly that internal, nondiffusive processes are limiting the speed of the reaction. The rate enhancement is due to an increase in the standard chemical potential of the ligand molecule as the polyalcohol concentration is increased. Both types of behavior are observed for ligand binding to protoheme in 0.1 N NaOH; first an increase and then a decrease in rate is observed as the concentration of glycerol is increased. At low glycerol concentrations, the reaction rate is limited by a first order process. At high concentrations, the rate becomes diffusion-controlled and exhibits a dependence on the reciprocal of the solvent viscosity. The data for all these conditions have been analyzed empirically in terms of a single free energy barrier and more specifically in terms of a consecutive reaction scheme.