Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.     

Changes in membrane and surface potential explain the opposite effects of low ionic strength on the two lysine transporters of human erythrocytes

The sucrose-induced stimulation of lysine influx in human erythrocytes has been attributed to the removal of a competitive inhibition exerted by Na+ on system y+ (Young, J. D., Fincham, D. A., and Harvey, C. M. (1991) Biochim. Biophys. Acta 1070, 111-118). We have reexamined this phenomenon separating the contribution of the two cationic amino acid transporters present in these cells (system y+ and system y+L). NaCl replacement with sucrose increased influx through system y+L, but decreased influx through system y+. We conclude that 1) the inhibition of system y+ is a response to the membrane depolarization that results from chloride removal, and 2) the stimulation of system y+L is due to the enhancement of the negative surface potential. Consistently, lysine influx through system y+L (in sucrose medium) was reduced by Na+, K+, Li+, and choline (K0.5 = 25-34 mM), the effect reaching a maximum at 35-40% of the original flux. Divalent cations (Ca2+ and Mg2+) were also inhibitory, but lower concentrations were required (K0.5 1.1-1.8 mM). The finding that sucrose stimulates uptake through changes in the surface potential explains similar effects observed in other cells with various cationic substrates.     

Effects of phase separating systems on lyophilized hemoglobin

Polymer liquid-liquid two-phase systems offer a unique opportunity to study the mechanisms of protein stabilization during freezing and freeze-drying. Fourier transform infrared spectroscopy was used to monitor the structural integrity of recombinant hemoglobin frozen and lyophilized in the separated phases of a polyethylene glycol (PEG)-dextran system. Protein in each phase of an equilibrated biphasic PEG-dextran system experiences similar levels of structural protection against freezing stresses despite large differences in polymer concentration. This result further demonstrates previous suggestions that proteins are protected during freezing by the preferential exclusion mechanism. There are, however, distinct differences in the level of structural protection that polymers in equilibrium phases provide to proteins during lyophilization, emphasizing that the mechanisms of protein protection during freezing and drying are fundamentally different. In addition, we provide evidence that phase separation per se occurring during the course of the lyophilization cycle can be detrimental to the structural stability of a protein.     

Free energy of burying hydrophobic residues in the interface between protein subunits

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379-400]. We determined by analytical ultracentrifugation the dimer-tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary alpha1 beta1 interface, and all of the others are at the sliding alpha1 beta2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the alphabeta dimer interface. The slope yields an interface stabilization free energy of -15 +/- 1.2 cal/mol upon burial of 1 A2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199-203].     

Deterministic pressure-induced dissociation of vicilin, the 7S storage globulin from pea seeds: effects of pH and cosolvents on oligomer stability

A thermodynamic characterization of subunit association of vicilin, a storage protein from pea seeds, was performed using a combination of hydrostatic pressure and fluorescence spectroscopy. Application of pressure up to 2.4 kbar caused dissociation of vicilin subunits, as revealed by (1) size-exclusion PPLC of pressurized samples, (2) fluorescence anisotropy measurements of a dansyl-vicilin conjugate under pressure, and (3) quenching of the intrinsic fluorescence of vicilin. Pressure dissociation data were well described by a model for dissociation of a trimer. This enabled calculation of the standard molar volume change of association (delta Vo) and the equilibrium dissociation constant at atmospheric pressure (Ko); at pH 10 these were found to be delta Vo = 146 mL/mol and Ko = 2.2 x 10(-15) M2, respectively, corresponding to C1/2 (the concentration of protein at 50% dissociation at atmospheric pressure) = 18 nM and a stabilization free energy of -19.6 kcal/mol for the oligomer. Vicilin exhibited an anomalously low dependence on protein concentration for pressure dissociation. This appeared related to conformational changes in the dissociated subunits, which caused a loss of ca. 5 kcal/mol in the free energy of association and to structural/energetic heterogeneity in the population of oligomers. Pressure dissociation was markedly pH-dependent, with a stabilization free energy loss of 3.4 kcal/mol upon raising pH from 9 to 10. Circular dichroism and intrinsic fluorescence lifetime measurements at atmospheric pressure showed that the structure of vicilin was largely unaffected by pH in the range investigated. These results suggest that the effect of pH may involve deprotonation of lysine residues participating in salt bridges between vicilin subunits. Pressure dissociation of vicilin was significantly inhibited by addition of salts (NaCl, KCl, LiCl) or glycerol. Dissociation curves obtained in the presence of salts enabled calculation of the free energies of stabilization (ranging from ca. -1.2 to -2.4 kcal/mol) of the vicilin oligomers by these cosolvents. The similar effects of salts or glycerol suggest a common mechanism of stabilization of the oligomer involving exclusion of the cosolvents from the protein interface and preferential hydration of the protein.     

Thermodynamic characterization of interactions of native bovine serum albumin with highly excluded (glycine betaine) and moderately accumulated (urea) solutes by a novel application of vapor pressure osmometry

The thermodynamic consequences of interactions of native bovine serum albumin (BSA) with two smaller solutes (glycine betaine or urea) in aqueous solution are characterized by a novel application of vapor pressure osmometry (VPO), which demonstrates the utility of this method of investigating preferential interactions involving solutes that are either accumulated or excluded near the surface of a protein. From VPO measurements of osmolality (water activity) as a function of the solute concentration in the presence and absence of BSA, we determine the dependence of the solute molarity (C3) on that of BSA (C2) at fixed temperature (37 degrees C), pressure (approximately 1 atm), and osmolality (over the range 0-1.6 molal). After some thermodynamic transformations, these results yield values of [formula: see text] which characterizes the interdependence of solute molalities when temperature, pressure, and the chemical potential of solute 3 are fixed. This form of the preferential interaction coefficient can be interpreted directly in terms of the molecular exclusion or accumulation of the solute (relative to water) near the protein surface. Within experimental uncertainty, [formula: see text] is proportional to m3 both for glycine betaine (0-0.9 m) and for urea (0-1.6 m). For glycine betaine [formula: see text] = -49 +/- 4, a value consistent with the interpretation that this solute is completely excluded from the hydrated surface of BSA, whereas for urea [formula: see text] = 6 +/- 1, which indicates a moderate extent of accumulation at the surface of native BSA. The preferential accumulation of solutes (e.g., urea) that have some binding affinity for a protein can be quantified and interpreted using the two-domain model if the extent of hydration of the protein has been determined using a completely excluded solute (e.g., glycine betaine). Complete exclusion from the local hydration domain surrounding proteins, if general, justifies the use of glycine betaine as a thermodynamic probe of the changes in hydration that accompany protein folding, protein association, and protein-ligand binding interactions.     

Size exclusion HPLC method for the determination of acidic fibroblast growth factor in viscous formulations

A size exclusion HPLC method has been developed to determine the protein concentration of pharmaceutical formulations of recombinant acidic fibroblast growth factor (aFGF). These topical aFGF formulations not only contain low levels of protein mass (50 micrograms ml-1), but also include buffer ions, polysaccharide polyanions to conformationally stabilize aFGF and 1% hydroxyethylcellulose to increase the solution's viscosity. A cesium chloride mobile phase is utilized during SEC-HPLC to dissociate aFGF from the pharmaceutical excipients and to minimize nonspecific interaction of the protein with the column matrix. The protein content of a viscous aFGF formulation is determined by comparison of aFGF peak areas to standards of known concentration. Fluorescence spectroscopy was utilized to directly demonstrate that the protein remains in its native conformation during sample preparation and analysis.     

A test of how well the repeatability of courtship predicts its heritability

In mossy fiber synapses of the hippocampal CA3 region, LTP is induced by cAMP and requires the synaptic vesicle protein rab3A. In contrast, CA1-region synapses do not exhibit this type of LTP. We now show that cAMP enhances glutamate release from CA3 but not CA1 synaptosomes by (1) increasing the readily releasable pool as tested by hypertonic sucrose; (2) potentiating release evoked by KCl depolarization, which opens voltage-gated Ca2+ channels; and (3) by enhancing Ca2+ action on the secretory apparatus as monitored by the Ca2+-ionophore ionomycin. In rab3A-deficient synaptosomes, forskolin still enhances KCl- and sucrose-induced glutamate release but not ionomycin-induced release. Our results show that cAMP has multiple actions in mossy fiber synapses, of which only the direct activation of the secretory apparatus requires rab3A and functions in mfLTP.     

Molecular cloning and functional expression of equine interleukin-1 receptor antagonist

Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and rabbit IL-1ras, respectively. An N-glycosylation site and five cysteine residues conserved in human, murine and rabbit IL-1ras were also found at the corresponding positions in equine IL-1ra. Recombinant glutathione S-transferase (GST)-equine IL-1ra fusion protein produced by Escherichia coli was purified. This protein was shown to inhibit the cytostatic or cytotoxic activity of IL-1 on A375S2 cells, indicating that the equine IL-1ra cDNA obtained in this study encodes biologically active equine IL-1ra.     

Energetics of inclusion-induced bilayer deformations

The material properties of lipid bilayers can affect membrane protein function whenever conformational changes in the membrane-spanning proteins perturb the structure of the surrounding bilayer. This coupling between the protein and the bilayer arises from hydrophobic interactions between the protein and the bilayer. We analyze the free energy cost associated with a hydrophobic mismatch, i.e., a difference between the length of the protein's hydrophobic exterior surface and the average thickness of the bilayer's hydrophobic core, using a (liquid-crystal) elastic model of bilayer deformations. The free energy of the deformation is described as the sum of three contributions: compression-expansion, splay-distortion, and surface tension. When evaluating the interdependence among the energy components, one modulus renormalizes the other: e.g., a change in the compression-expansion modulus affects not only the compression-expansion energy but also the splay-distortion energy. The surface tension contribution always is negligible in thin solvent-free bilayers. When evaluating the energy per unit distance (away from the inclusion), the splay-distortion component dominates close to the bilayer/inclusion boundary, whereas the compression-expansion component is more prominent further away from the boundary. Despite this complexity, the bilayer deformation energy in many cases can be described by a linear spring formalism. The results show that, for a protein embedded in a membrane with an initial hydrophobic mismatch of only 1 A, an increase in hydrophobic mismatch to 1.3 A can increase the Boltzmann factor (the equilibrium distribution for protein conformation) 10-fold due to the elastic properties of the bilayer.     

The folded conformation of phage P22 coat protein is affected by amino acid substitutions that lead to a cold-sensitive phenotype

Three cold-sensitive mutants in phage P22 coat protein have been characterized to determine the effects of the amino acid substitutions that cause cold sensitivity on the folding pathway and the conformation of refolded coat protein. Here we find that the three cold-sensitive mutants which have the threonine residue at position 10 changed to isoleucine (T10I), the arginine residue at position 101 changed to cysteine (R101C), or the asparagine residue at position 414 changed to serine (N414S) were capable of folding from a denatured state into a soluble monomeric species, but in each case, the folded conformation was altered. Changes in the kinetics of folding were observed by both tryptophan and bisANS fluorescence. In contrast to the temperature-sensitive for folding coat protein mutants which can be rescued at nonpermissive temperatures in vivo by the overproduction of molecular chaperones GroEL and GroES [Gordon, C. L., Sather, S. K., Casjens, S., & King, J. (1994) J. Biol. Chem. 269, 27941-27951], the folding defects associated with the cold-sensitive amino acid substitutions were not recognized by GroEL and GroES.     

A transient expansion of the native state precedes aggregation of recombinant human interferon-gamma

Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-gamma (rhIFN-gamma) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-gamma aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-gamma and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-gamma native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-gamma against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.     

Thermodynamics of apocytochrome b5 unfolding

Apocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two-state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change at protein unfolding (delta G) amounts to delta G = 7 +/- 1 kJ/mol at 25 degrees C (pH 7.4) compared with delta G = 25 kJ/mol for the holoprotein. Apocytochrome b5 is a compact, native-like protein. According to the spectral data, the cooperative structure is mainly based in the core region formed by residues 1-35 and 79-90. This finding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J., 1993, Biochemistry 32, 199-207).     

Gloverin, an antibacterial protein from the immune hemolymph of Hyalophora pupae

Gloverin is an inducible antibacterial insect protein isolated from pupae of the giant silk moth Hyalophora. It is a basic (pI 8.5) protein with a molecular mass of 13.8 kDa, containing a large number of glycine residues (18.5%) but no cysteine, and has an amino acid sequence that reveals no strong degree of identity with any known proteins. Gloverin inhibits the growth of Escherichia coli at a minimal concentration of 1-3 microM, i.e. less than 5% of the concentration of gloverin in the hemolymph of infected pupae. The prime effect of gloverin, following its interaction with lipopolysaccharide (LPS) in the bacterial envelope, is a specific inhibition of the synthesis of vital outer membrane proteins, leading to an increased permeability of the outer membrane. The activity of gloverin is not affected by heating (100 degrees C, 10 min) but is inhibited by Mg2+ and by free LPS. The gloverin molecule will undergo conformational transitions from a monomeric random coil to an alpha-helix upon transfer from an aqueous to a hydrophobic environment, a property likely to be of importance for its interaction with cell-bound LPS. The activity of gloverin is in many respects similar to that of attacin, another antibacterial protein, originally found in Hyalophora [for a review see Boman, H. G., Faye, I., Gudmundsson, G. H., Lee, J.-Y. & Lindholm, D. A. (1991) Eur J. Biochem. 201, 23-31].     

NMR solution structure of the oxidized form of MerP, a mercuric ion binding protein involved in bacterial mercuric ion resistance

Mercuric ions are toxic to living organisms because of their strong affinity for cysteine residues in proteins. Some bacteria have developed a resistance mechanism whereby Hg2+ is transported into the cytoplasm and reduced to Hg0. One of the proteins involved in the transport of mercuric ion is the periplasmic binding protein MerP, which can exist both as oxidized (disulfide) and as reduced (dithiol) forms. Only the reduced form with Cys-17 and Cys-14 residues as free thiols is a potent receptor for mercuric ion. In this work the solution structure of the oxidized form of MerP has been determined by multidimensional NMR spectroscopy and compared to the NMR structures of the previously published structures of the reduced and mercury-bound forms of MerP. The mercury-bound and oxidized forms have similar tertiary structures, whereas in the reduced form there is a large rearrangement of the mercuric ion binding loop and the nearby loop comprising residues 38-41. The structural arrangement of the latter loop seems to be important for the stabilization of the surface location of the cysteine-containing loop. In the reduced form at low pH the cysteine-containing loop adopts a conformation similar to what is observed in the oxidized and mercury-bound forms. The oxidized form also differs with respect to the other two forms in the relative positions of some of the alpha-helices and beta-strands. Structural differences between the oxidized and reduced forms may help explain why the reduced form is stable in the periplasm, which is considered to be an oxidizing environment.     

A phase II study of high-dose epirubicin (EPI) plus cyclophosphamide (CPA) with G-CSF for breast cancer patients with visceral metastases or hormone-independent tumors: a trial of the Japan Clinical Oncology Group

In a previous study, we found that oral chromium nicotinate overcame sucrose-induced hypertension in spontaneously hypertensive rats (SHR). Accordingly, we examined more chromium compounds to determine if others were more or less effective in regulating blood pressure (BP) of SHR. Since chromium is postulated to be an antioxidant, we also assessed the ability of different chromium compounds to alter free radical formation measured by determining thiobarbituric acid reactive substances (TBARS). The control group of SHR ingested a diet low in chromium, and 5 other groups ate the same diet with various chromium compounds added at 5 ppm-chloride, acetate, nicotinic acid-glycine-cysteine-glutamic acid (NA-AA), picolinate, and nicotinate. Following this, the rats were challenged with drinking water containing 5% and 10% w/v sucrose. Except for NA-AA, all chromium compounds inhibited the sucrose-induced elevation of systolic BP; and acetate, picolinate, and nicotinate chromium compounds lowered HbAIC below control. Only chromium acetate and nicotinate significantly lowered both hepatic and renal TBARS. Chromium picolinate lowered hepatic TBARS, and chromium chloride and NA-AA lowered neither. We conclude that chromium, rather than a specific ligand, plays a major role in ameliorating sucrose-induced BP elevations and can act as an antioxidant.     

Improved folding yields of a model protein using protein disulfide isomerase

PURPOSE: To study the effects of recombinant human protein disulfide isomerase (rhPDI) concentration, reduced glutathione:oxidized glutathione ratio (GSH:GSSG) and temperature on the efficiency of oxidative folding of a model protein, recombinant human interleukin 2 (C125A mutation) (C125A rhIL-2). METHODS: C 125A rhIL-2 inclusion bodies were reduced and denatured by guanidium hydrochloride (Gdm.Cl) and 100 mM GSH. The solution was diluted 10 times into folding buffer, allowing C125A rhIL-2 to fold either in the absence or presence of rhPDI. The renatured and unfolded C125A rhIL-2 species were quantitated by reversed phase-HPLC. RESULTS: The initial folding rate of C125A rhIL-2 linearly increased with rhPDI:C125A rhIL-2 molar ratio in the first 2.5 minutes, and reached the highest rate when the rhPDI:C125A rhIL-2 ratio was 1:1. The oxidative folding of C125A rhIL-2 linearly increased as the GSH:GSSG molar ratio decreased from 10:0 to 10:3. The folding of C125A rhIL-2 was also dependent on temperature, and optimum folding was realized at 23 degrees C. CONCLUSIONS: These results demonstrate that under optimal redox potential and temperature, rhPDI enhances the oxidative folding of C125A rhIL-2. In the oxidative folding of C125A rhIL-2, rhPDI exerts its effect on folding by the acceleration of thiol/disulfide interchange.     

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A, Mg2+ Ap5A, and Mn2+ Ap5A reveal an intermediate lid position and six coordinate octahedral geometry for bound Mg2+ and Mn2+

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A, Mn2+ Ap5A, and Mg2+ Ap5A have been determined by X-ray crystallography to resolutions of 1.6 A, 1.85 A, and 1.96 A, respectively. The protein's lid domain is partially open, being both rotated and translated away from bound Ap5A. The flexibility of the lid domain in the ternary state and its ability to transfer force directly to the the active site is discussed in light of our proposed entropic mechanism for catalytic turnover. The bound Zn2+ atom is demonstrably structural in nature, with no contacts other than its ligating cysteine residues within 5 A. The B. stearothermophilus adenylate kinase lid appears to be a truncated zinc finger domain, lacking the DNA binding finger, which we have termed a zinc knuckle domain. In the Mg2+ Ap5A and Mn2+ Ap5A structures, Mg2+ and Mn2+ demonstrate six coordinate octahedral geometry. The interactions of the Mg2+-coordinated water molecules with the protein and Ap5A phosphate chain demonstrate their involvement in catalyzing phosphate transfer. The protein selects for beta-y (preferred by Mg2+) rather than alpha-gamma (preferred by Mn2+) metal ion coordination by forcing the ATP phosphate chain to have an extended conformation.     

The C-terminal domain of matrilin-2 assembles into a three-stranded alpha-helical coiled coil

Matrilin-2 is a member of von Willebrand factor A containing extracellular matrix proteins in which the cDNA-derived sequence shows similar domain organization to cartilage matrix protein/matrilin-1, but information on the protein structure is limited. Here we studied the oligomerization potential of a synthetic peptide NH2-ENLILFQNVANEEVRKLTQRLEEMTQRMEALENRLKYR-COOH corresponding to the C-terminal sequence of mouse matrilin-2. The central portion of this sequence shows a periodicity of hydrophobic residues occupying positions a and d of a heptad pattern (abcdefg)n, which is characteristic for alpha-helical coiled-coil proteins. Circular dichroism spectroscopy revealed a high alpha-helical content, and the shape of the spectra is indicative for a coiled-coil conformation. Chemical cross-linking and size exclusion chromatography suggest a homotrimeric configuration. Thermal denaturation in benign buffer shows a single cooperative transition with DeltaH0 = -375 kJ/mol. Melting temperatures Tm varied from 38 to 51 degreesC within a concentration range of 10 to 85 microM, which is about 35 degreesC lower than determined for a peptide corresponding to the C-terminal domain of matrilin-1. The data suggest that despite the low sequence identity within this region, matrilin-2 will form a homotrimer as matrilin-1 does.