Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: evidence of a reversible acid-denatured molten globule.

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation. A reversible acid-denatured molten globule state was observed from circular dichroism and fluorescence spectroscopy data. The molten globule state was characterized by a loss of tertiary structure with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resulting in approximately 90% recovery of the native structure. The molten globule state was characterized by a broadening of structural features in the second-derivative spectra of Fourier transform infrared spectroscopy. A component band at 1650 cm(-1) was shown to be alpha-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorimetry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanying baseline shift at 58 degrees C (a consequence of increased heat capacity). The tendency to form molten globules during acid denaturation stress permits an opportunity to study the process of partial protein unfolding with implications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten globule state is formed exclusively within the beta-sheet receptor-binding region.

An antiparallel four-helix bundle orients the high-affinity RNA binding sites in hnRNP C: a mechanism for RNA chaperonin activity.

Previous studies have shown that the C protein of 40 S hnRNP complexes contains a leucine-zipper domain, residues 180-207, and that a 40 residue highly basic domain, immediately preceding the zipper, is responsible for almost all of the free energy of RNA binding to C protein. Because this domain arrangement is like that seen in the bZIP transcription factors it has been termed the bZIP-like-motif or bZLM. We report here that the zipper domain drives C protein oligomerization through its spontaneous assembly into an anti-parallel four-helix bundle approximately 50 A in length. The anti-parallel nature of the four-helix bundle positions the tetramer's four high-affinity RNA binding domains at opposing ends of a rigid core formed by the helix bundle. This domain topology is ideally suited to accommodate and direct a double wrapping of RNA around the tetramer and is fully consistent with C protein's ability to bind and order 230 nt lengths of pre-mRNA through a highly cooperative RNA binding mode. We have used a novel sequence-specific 13C/15N labeling strategy and multidimensional NMR spectroscopy to define the anti-parallel orientation of the four-helix bundle and its molecular dimensions. In vitro reconstitution and hydrodynamic studies on native C protein, on several C protein fragments, and on various synthetic peptides, are consistent with the proposed model and indicate that C protein's canonical RNA recognition motifs probably function in tetramer-tetramer interactions during 40 S hnRNP assembly.

SecA folds via a dimeric intermediate.

Though many proteins in the cell are large and multimeric, their folding has not been extensively studied. We have chosen SecA as a folding model because it is a large, homodimeric protein (monomer molecular mass of 102 kDa) with multiple folding domains. SecA is the ATPase for the Sec-dependent preprotein translocase of many bacteria. SecA is a soluble protein that can penetrate into the membrane during preprotein translocation. Because SecA may partially unfold prior to its insertion into the membrane, studies of its stability and folding pathway are important for understanding how it functions in vivo. Kinetic folding transitions in the presence of urea were monitored using circular dichroism and tryptophan fluorescence, while equilibrium folding transitions were monitored using the same techniques as well as a fluorescent ATP analogue. The reversible equilibrium folding transition exhibited a plateau, indicating the presence of an intermediate. Based on the data presented here, we propose a three-state model, N(2) if I(2) if 2U, where the native protein unfolds to a dimeric intermediate which then dissociates into two unfolded monomers. The SecA dimer was determined to have an overall stability (DeltaG) of -22.5 kcal/mol. We also investigated the stability of SecA using analytical ultracentrifugation equilibrium and velocity sedimentation, which again indicated that native or refolded SecA was a stable dimer. The rate-limiting step in the folding pathway was conversion of the dimeric intermediate to the native dimer. Unfolding of native, dimeric SecA was slow with a relaxation time in H(2)O of 3.3 x 10(4) s. Since SecA is a stable dimer, dissociation to monomeric subunits during translocation is unlikely.

Ultracentrifuge and circular dichroism studies of folding equilibria in a retro GCN4-like leucine zipper.

Equilibrium ultracentrifuge and circular dichroism (CD) studies of a retropeptide of a GCN4-like leucine zipper in neutral saline buffer are reported as functions of temperature. Ultracentrifuge results indicate the presence of three oligomeric species: monomer, dimer, and tetramer, in quantifiable amounts, and the data provide values for the standard DeltaG, DeltaH, and DeltaS for interconversion. CD at 222 nm displays the strong concentration dependence characteristic of dissociative unfolding, but also shows a helicity far below that of the parent propeptide. Remarkably enough, the CD at 222 nm shows an extremum in the region between 0 and 20 degrees C. At higher T, the usual cooperative unfolding is observed. Comparable data are presented for a mutant retropeptide, in which a single asparagine residue is restored to the characteristic heptad position it occupies in the propeptide. The mutant shows marked differences from its unmutated relative in both thermodynamic properties and CD, although the oligomeric ensemble also comprises monomers, dimers, and tetramers. The mutant is closer in helicity to the parent propeptide but is less stable. These findings do not support either of the extant views on retropeptides. The behavior seen is consistent neither with the view that retropeptides should have the same structure as propeptides nor with the view that they should have the same structure but opposite chirality. The simultaneous availability of oligomeric population data and CD allows the latter to be dissected into individual contributions from monomers, dimers, and tetramers. This dissection yields explanations for the observed extrema in curves of CD (222 nm) versus T and reveals that the dimer population in both retropeptides undergoes "cold denaturation."

Energetics of assembling an artificial heterodimer with an alpha/beta motif: cleaved versus uncleaved Escherichia coli thioredoxin.

We have studied the folding/binding process between the N- and C-fragments (1-73, 74-108) of oxidized Escherichia coli thioredoxin (Trx) to compare the energetics between the cleaved and uncleaved Trx. Sedimentation equilibrium analysis in 0.1 M potassium phosphate, pH 5.7, shows (i) the strong and weak self-association of the N- and C-fragments, respectively, (ii) a heterodimer with a small dissociation constant (K(d)) ca. 100 nM, and (iii) monomeric Trx. To avoid self-association, measurements were carried out in 10 mM potassium phosphate, pH 5.7. Far-UV CD spectra of the fragments at variable temperature show an isodichroic point at 208 nm and a non-cooperative cold induced disordering transition without concentration dependence. Deconvolution of these spectra indicates the presence of residual structure. Titration of the N-fragment with an excess of C-fragment indicates a 1:1 stoichiometric complex with an apparent K(d) ca. 49 nM. Analysis of this complex by CD and hydrogen exchange/2D-NMR (Tasayco and Chao (1995) Proteins: Struct., Funct., Genet. 22, 41-44) spectroscopy indicates the reassembly of the alpha/beta motif of Trx. GnHCl induced unfolding measurements give DeltaG(0) values of 9.5 +/- 0.2 and 10.0 +/- 0.4 kcal/mol at 20 degrees C for the uncleaved and cleaved Trx, respectively. The far-UV CD melting curve of uncleaved Trx indicates an intriguing non-cooperative upward baseline trend. CCA analysis of these spectra indicates the presence of a native-like folded intermediate. A three-state thermodynamic analysis of the thermal transition curves gives a total DeltaH(0) of unfolding of 121 +/- 4 kcal/mol at the T(m) (88 degrees C), while the two-state analysis for cleaved Trx gives 122 +/- 6 kcal/mol at 88 degrees C. Analysis of the chemical and thermal unfolding of both proteins indicates a value of ca. 1 M for the apparent effective concentration (C(eff)) of cleaved Trx.

Characterization of the dimer interface of transcription factor NFkappaB p50 homodimer.

Dimers of the Rel/NFkappaB transcription factor family form with differential stabilities through the combinatorial association of five polypeptides: p50, p52, p65, cRel, and RelB. Here, we have characterized the nature of the monomer-dimer equilibrium of the p50 homodimer. Sedimentation equilibrium studies show that the equilibrium constant for p50 dimer dissociation is in the low micromolar range. Using the X-ray crystal structure of the p50 homodimer as a guide, we have created site-directed alanine mutations at ten dimer-forming residues in p50 and measured their effects on p50 homodimerization. Characterization of these alanine mutants by a series of chemical crosslinking, size-exclusion chromatography, and sedimentation equilibrium experiments shows that the most critical residue in stabilizing the p50 dimer interface is Y267. Sedimentation equilibrium experiments show that an alanine substitution at position 267 destabilizes the dimer interface by 2.0 kcal/mol. Alanine substitutions at two other positions, L269 and V310, significantly destabilize the p50 dimer interface. These two residues are observed to mediate critical interactions in the crystal structure. Together, these three residues constitute the "hot-spot" of protein-protein interaction in p50 dimerization. Of the four charged residues in the dimer interface, R252, D254, E265, and D302, only D302 contributes significantly to p50 dimer stability. D254 appears to slightly destabilize the subunit interface. Although residues H304, R305, and F307 occupy positions at the hydrophobic core of the interface and appear to be involved in multiple interactions in the X-ray crystal structure, alanine substitutions at these positions do not significantly reduce the affinity for p50 dimerization. Upon evaluating the roles of these amino acid residues at the p50 dimer interface, we propose that differential contributions of a few key residues dictate the selectivity of dimer formation within the Rel/NFkappaB family.

Dissection of the de novo designed peptide alpha t alpha: stability and properties of the intact molecule and its constituent helices.

Alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in solution [Fezoui et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3675-3679; Fezoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. alpha t alpha was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The Delta G0 of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant for folding of alpha t alpha was estimated from the Delta G0 as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the component peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.

Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris.

The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus.

Structural and biological stability of the human interleukin 10 homodimer.

Human interleukin 10 (huIL-10) is a cytokine that regulates the synthesis of type 1 helper T cell derived cytokines such as gamma-interferon, interleukin 2, and tumor necrosis factor alpha. The potential immunosuppressive activities of huIL-10 suggest that this protein may be clinically useful for treating autoimmune diseases. Due to the potential clinical value of this cytokine, physicochemical studies have been performed regarding its association state and biological/structural stability. These studies include performing size-exclusion chromatography, chemical cross-linking, equilibrium ultracentrifugation, and circular dichroism spectroscopy. The results indicate huIL-10 is predominantly a noncovalent homodimer at neutral pH and 4 degreesC for concentrations greater than 0.003 mg/mL (0.08 microM dimer). An apparent pKa value of approximately 4.8 was calculated for both the pH-dependent subunit dissociation and pH-induced loss in MC/9 biological activity. A temperature analysis revealed a linear relationship between the percent dimer and relative MC/9 activity, thus, these results and the pH-dependent activity results suggest that the huIL-10 dimer is the active species. The GndHCl-induced unfolding of rhuIL-10, monitored by far-UV circular dichroism, revealed a unique biphasic unfolding process which contained both a subunit dissociation process (<1.6 M GndHCl) as well as the unfolding of a highly alpha-helical monomer intermediate ([GndHCl]1/2 = 3.5 M). The monomer intermediates generated with 1.6 M GndHCl or pH 2.5 retained approximately 80% and 89% of the alpha-helical content of the native protein, respectively. Although a soluble and highly helical monomer state can be generated, the observed correlation between unfolding studies and biological activity suggests the dimer is the active species. These results are consistent with both the recent observation that the three-dimensional structure of rhuIL-10 is a 2-fold symmetric homodimer and that a complex between the extracellular domain of the recombinant human IL-10 receptor and IL-10 is consistent with two IL-10 homodimers and four receptors.

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein.

MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell division site of Escherichia coli. We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gels. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE22-88 fragment forms hetero-oligomers with MinE+ when the proteins are co-expressed. In contrast, the MinE36-88 fragment does not form MinE+/MinE36-88 hetero-oligomers, although MinE36-88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE+ in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of minicelling by expression of MinE36-88 in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE36-88 and the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.

Recombinant soluble alphabeta T cell receptors protect T cells from immune suppression: requirement for aggregated multimeric, disulfide-linked alphabeta heterodimers.

Recombinant soluble T cell receptors (sTCR) protected contact sensitivity (CS) effector T cells from down-regulation or immunosuppression. CS-protecting sTCR were released enzymatically from the surface of thymoma cells transfected with cDNAs encoding TCR-alpha and -beta extracellular domains that were expressed with a phosphatidylinositol linkage. sTCR affinity purified on anti-TCR-alpha and anti-TCR-beta mAb columns had identical CS-protective activity, as did sTCR from a CD4+ Th2 clone or from a CD8+ cytotoxic clone. Reduced sTCR alpha- and beta-chains had no CS-protective activity, but this was restored when the TCR chains were rejoined into disulfide-linked alphabeta heterodimers. sTCR CS protection was Ag nonspecific, MHC unrestricted, and not influenced by the relevant synthetic peptide specific for the TCR complexed with appropriate MHC. CS protection may have resided in the sTCR constant region. When heated at 62 degrees C for 30 min, sTCR formed a CS-protecting aggregate, with a molecular mass of 481 +/- 37 kDa, corresponding to an alphabeta TCR pentamer. HPLC gel filtration essentially confirmed the molecular mass at 516 kDa for the multimer, while the monomer, which was an alphabeta TCR heterodimer, had an expected molecular mass of approximately 104 kDa and no bioactivity. In summary, the pentameric sTCR may bind to and activate lymphoid cells, perhaps via constant domains, resulting in protection of CS effector T cells from down-regulation. The ability of sTCR to protect CS effector T cells from down-regulation/suppression, if generalized, could overcome immunosuppression accompanying infectious diseases, particularly AIDS, or in tumors.

Ligand linked assembly of Scapharca dimeric hemoglobin.

The assembly of Scapharca dimeric hemoglobin as a function of ligation has been explored by analytical gel chromatography, sedimentation equilibrium, and oxygen binding experiments to test the proposal that its cooperativity is based on quaternary enhancement. This hypothesis predicts that the liganded form would be assembled more tightly into a dimer than the unliganded form and that dissociation would lead to lower oxygen affinity. Our experiments demonstrate that although the dimeric interface is quite tight in this hemoglobin, dissociation can be clearly detected in the liganded states with monomer to dimer association constants in the range of 10(8) M-1 for the CO-liganded state and lower association constants measured in the oxygenated state. In contrast, the deoxy dimer shows no detectable dissociation by analytical ultracentrifugation. Thus, the more highly hydrated deoxy interface of this dimer is also the more tightly assembled. Equilibrium oxygen binding experiments reveal an increase in oxygen affinity and decrease in cooperativity as the concentration is lowered (in the muM range). These experiments unambiguously refute the hypothesis of quaternary enhancement and indicate that, as in the case of human hemoglobin and other allosteric proteins, quaternary constraint underlies cooperativity in Scapharca dimeric hemoglobin.

Binding of L-branched-chain amino acids causes a conformational change in BkdR.

BkdR is the positive transcriptional activator of the inducible bkd operon of Pseudomonas putida. Evidence is accumulating that L-branched-chain amino acids are the inducers of the operon, and the data obtained in this study show that they induce a conformational change in BkdR. Addition of L-branched-chain amino acids increased the susceptibility of BkdR to trypsin with the cleavage between Arg-51 and Gln-52 on the C-terminal side of the DNA-binding domain. L-Valine also caused an increased fluorescence emission intensity and produced significant changes in the circular dichroism spectrum of BkdR. Analytical ultracentrifugation confirmed earlier data obtained from gel filtration that BkdR was a tetramer with a Stokes radius of 32 +/- 3 A and an axial ratio of 2:1.

Duplex-tetraplex equilibrium between a hairpin and two interacting hairpins of d(A-G)10 at neutral pH.

d(A-G)10 forms two helical structures at neutrality, at low ionic strength a single-hairpin duplex, and at higher ionic strength a double-hairpin tetraplex. An ionic strength-dependent equilibrium between these forms is indicated by native PAGE, which also reveals additional single-stranded species below 0.3 M Na+, probably corresponding to partially denatured states. The equilibrium also depends upon oligomer concentration: at very low concentrations, d(A-G)10 migrates faster than the random coil d(C-T)10, probably because it is a more compact single hairpin; at high concentrations, it co-migrates with the linear duplex d(A-G)10 x d(C-T)10, probably because it is a two-hairpin tetraplex. Molecular weights measured by equilibrium sedimentation in 0.1 M Na+, pH 7, reveal a mixture of monomer and dimer species at 1 degree C, but only a monomer at 40 degrees C; in 0.6 M Na+, pH 7, only a dimer species is observed at 4 degrees C. That the single- and double-stranded species are hairpin helices, is indicated by preferential S1 nuclease cleavage at the center of the oligomer(s), i.e., the loop of the hairpin(s). The UV melting transition below 0.3 M Na+ or K+, exhibits a dTm/dlog[Na+/K+] of 33 or 36 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex to a single-hairpin duplex with extrahelical residues. When [Na+/K+] > or = 0.3 M, dTm/dlog [Na+/K+] is 19 or 17 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex directly to single strands. A two-hairpin structure stabilized by G-tetrads is indicated by differential scanning calorimetry in 0.15 M Na+/5 mM Mg2+, with deltaH of formation per mole of the two-hairpin tetraplex of -116.9 kcal or -29.2 kcal/mol of G-tetrad.

Molecular shape, dissociation, and oxygen binding of the dodecamer subunit of Lumbricus terrestris hemoglobin.

Small angle x-ray scattering of the 213-kDa dodecamer of Lumbricus terrestris Hb yielded radius of gyration = 3.74 +/- 0.01 nm, maximum diameter = 10.59 +/- 0.01 nm, and volume = 255 +/- 10 nm3, with no difference between the oxy and deoxy states. Sedimentation velocity studies indicate the dodecamer to have a spherical shape and concentration- and Ca2+-dependent equilibria with its constituent subunits, the disulfide-bonded trimer of chains a-c and chain d. Equilibrium sedimentation data were fitted best with a trimer-dodecamer model, ln K4 = 7 (association K in liters3/g3) at 1 degrees C and 4 at 25 degrees C, providing DeltaH = -20 kcal/mol and DeltaS = 4.4 eu/mol. Oxydodecamer dissociation at pH 8.0, in urea, GdmCl, heteropolytungstate K8[SiW11O39] and of metdodecamer at pH 7, was followed by gel filtration. Elution profiles were fitted with exponentially modified gaussians to represent the three peaks. Two exponentials were necessary to fit all the dissociations except in [SiW11O39]-8. Equilibrium oxygen binding measurements at pH 6.5-8. 5, provided P50 = 8.5, 11.5-11.9 and 11.9-13.5 torr, and n50 = 5.2-9. 5, 3.2-4.9, and 1.8-2.7 for blood, Hb, and dodecamer, respectively, at pH 7.5, 25 degrees C. P50 was decreased 3- and 2-fold in approximately 100 mM Ca2+ and Mg2+, respectively, with concomitant but smaller increases in cooperativity.

Observation via one-dimensional 13Calpha NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper.

Synthesis of a 33-residue, capped leucine zipper analogous to that in GCN4 is reported. Histidine and arginine residues are mutated to lysine to reduce the unfolding temperature. CD and ultracentrifugation studies indicate that the molecule is a two-stranded coiled coil under benign conditions. Versions of the same peptide are made with 99% 13Calpha at selected sites. One-dimensional 13C NMR spectra are assigned by inspection and used to study thermal unfolding equilibria over the entire transition from 8 to 73 degrees C. Spectra at the temperature extremes establish the approximate chemical shifts for folded and unfolded forms at each labeled site. Resonances for each amino acid appear at both locations at intermediate T, indicating that folded and unfolded forms interconvert slowly (> >2 ms) on the NMR time scale. Moreover, near room temperature, the structured form's resonance is double at several, but not all, sites, indicating at least two slowly interconverting, structured, local conformational substates. Analysis of the dynamics is possible. For example, near room temperature at the Val-9, Ala-24, and Gly-31 positions, the equilibrium constant for interconversion of the two structured forms is near unity and the time scale is > or= 10-20 ms.

Mass spectrometric composition and molecular mass of Lumbricus terrestris hemoglobin: a refined model of its quaternary structure.

Sedimentation equilibrium measurements and scanning transmission electron microscopy (STEM) mass mapping of the extracellular, hexagonal bilayer hemoglobin (HBL Hb) of the earthworm Lumbricus terrestris provided masses of 3.41 to 3.66 MDa and 3.56 (+/- 0.13) MDa, respectively. The Hb also contains 57.2 (+/- 6.0) moles of tightly bound Ca per mole of protein. The Hb and its subunits obtained by dissociation, in native, dehemed and reduced carbamidomethylated forms, were subjected to electrospray ionization mass spectroscopy (ESI-MS). Maximum entropy deconvolution identified three groups of peaks, at approximately 16 kDa, 24 to 32 kDa and approximately 53 kDa corresponding to the monomer subunit M (globin chain d), four linker subunits and the disulfide-bonded trimer T (globin chains a + b + c). Subunit M consisted of three components, d1 (15, 992.4), d2 (15, 978.0) and d3 (15, 962.1) (+/- 1.0 Da), with relative intensities 1.0:5:0.3, respectively. Subunit T consisted of four major components, T1 (52, 922.6), T2 (52, 760.0), T3 (52, 598.5) and T4 (52, 435.4) (+/- 4.0 Da), with relative intensities 0.6:1.0:0.2:0.7, respectively. ESI-MS of carbamidomethylated T, demonstrated that, unlike chains b (16, 254.4) and c (17, 289.2), chain a exists as a series of four, hexose-connected, glycosylated isoforms, a1 to a4 (19, 389.9, 19, 227.4, 19, 065.3 and 18, 902.9) (+/- 1.0 Da). The mass differences between the deglycosylated chain a (17, 524.0) and a1 to a4 correspond to glycan side-chains (GlcNAc)2 (Man)n (n = 6 to 9). Four groups of peaks were observed in the 24 to 32 kDa region. Linkers L1a (27, 540.8) and L1b (27, 702.4) (+/- 2.0 Da) are isoforms of L1 (25, 837.5 in N-deglycosylated Hb) with glycan side-chains (GlcNAc)2 (Man)n (n = 8,9). Linkers L2 (32, 104.3 (+/- 5.0) Da) and L3 (24, 912.9 (+/- 2.0) Da) occur as single species. Linkers L4a to L4c (24, 019.0, 24, 102.3 and 24, 169.9) (+/- 2.0 Da) with relative intensities 1.0:0.8:0.8, have not been identified previously. From ESI-MS relative intensities, L1:L2:L3:L4 = 0.6:0.4:1.0:0.5 and globin linker = 0.78:0.22. HPLC of Lumbricus Hb provided a globin linker = 0.73:0.27 (+/- 0.02) and a heme content of 2.52 (+/- 0.14) wt%. A model is proposed for the HBL structure, wherein 12 213.4 kDa dodecamers (144 globin chains, 2561 kDa) decorate a hexagonal framework of 36 linker chains (12L1 + 6L2 + 12L3 + 6L4) to provide a total mass of 3.531 MDa, each dodecamer being in contact with three linker subunits.

Purification of active E1 alpha 2 beta 2 of Pseudomonas putida branched-chain-oxoacid dehydrogenase.

Active E1 component of Pseudomonas putida branched-chain-oxoacid dehydrogenase was purified from P. putida strains carrying pJRS84 which contains bkdR (encoding the transcriptional activator) and bkdA1 and bkdA2 (encoding the alpha and beta subunits). Expression was inducible, however, 45-, 39- and 37-kDa proteins were produced instead of the expected 45-kDa and 37-kDa proteins. The 45-kDa protein was identified as E1 alpha and the 37-kDa and 39-kDa proteins were identified as separate translational products of bkdA2 by their N-terminal sequences. The N-terminal amino acid of the 39-kDa protein was leucine instead of methionine. The 45-, 39- and 37-kDa proteins were also produced in wild-type P.putida. Translation of bkdA1 and bkdA2 from an Escherichia coli expression plasmid produced only 45-kDa and 39-kDa proteins, with N-terminal methionine on the 39-kDa protein. The insertion of guanine residues 5' to the first ATG of bkdA2 did not affect expression of E1 beta in P. putida including the N-terminal leucine which appears to eliminate the possibility of ribosome jumping. The Z-average molecular mass of the E1 component was determined by sedimentation equilibrium to be 172 +/- 9 kDa compared to a calculated value of 166 kDa for the heterotetramer and a Stokes radius of 5.1 nm. E1 alpha Ser313, which is homologous to the phosphorylated residue of rat liver E1 alpha, was converted to alanine resulting in about a twofold increase in Km, but no change in Kcat. S315A and S319A mutations had no effect on Km or Kcat indicating that these residues do not play a major part in catalysis of E1 alpha beta 2.

Mass spectrometric composition, molecular mass and oxygen binding of Macrobdella decora hemoglobin and its tetramer and monomer subunits.

The hexagonal bilayer hemoglobin (Hb) of the leech Macrobdella decora has an equilibrium sedimentation mass of 3544(+/- 80) kDa. Maximum entropy analysis of the electrospray ionization mass spectra of the Hb show three groups of peaks: two peaks of equal intensity at approximately 17 kDa, A (16,770.1 Da) and B (16,841.9 Da); three peaks at approximately 24 kDa, C (24,340.1 Da), D (24,398.6 Da) and E (24,420.0 Da) with relative intensities of 1:6:3, respectively; and three peaks of equal intensities at approximately 33 kDa, F (32,586.0 Da), G (32,714.5 Da) and H (32,849.9 Da). Although reduction with dithiothreitol does not affect the masses of peaks A through E, the approximately 33 kDa peaks give rise to four new peaks at approximately 16 kDa, P (16,052.2 Da), Q (16,537.3 Da), R (16,666.7 Da) and S (16,792.9 Da), indicating that F, G and H represent disulfide-bonded dimers of globin chains, P + Q, P + R and P + S, respectively. The relative intensities of the three groups of peaks are (A + B) to (C + D + E) to (F + G + H) = 0.39:0.26:0.32, and the globin to linker ratio 0.71:0.29 is in good agreement with the ratio 0.72:0.28 obtained by HPLC. The largest functional subunit obtained by dissociation at pH 7 in 4 M urea, is a subunit lacking linker chains with apparent mass 63(+/- 3) kDa. The equilibrium sedimentation profile of this subunit is fitted best as a monomer-dimer-tetramer equilibrium, with association constants K1,2 = 365 l g-1 and K1,4 = 8.1 x 10(5) l3 g-3. A model of the Hb consisting of a hexagonal bilayer of 36 tetramer and 42 linker subunits provides a total mass and globin to linker ratio closest to the experimental values. Equilibrium O2 binding measurements of the native Hb and its tetramer and monomer subunits were carried out over the pH range 6.6 to 8.0 at 10 and 25 degrees C, and in the absence and presence of Na+, Mg2+ and Ca2+. The Hb exhibits a moderately high O2 affinity, P50 = 4.4 torr at pH 7.5 and 25 degrees C, a high cooperativity (n50 approximately 3) and a substantial Bohr effect, phi = delta log P50/delta pH = -0.38. The tetramer subunit has a higher affinity, lower cooperativity and smaller Bohr effect, 1.9 torr, 1.3 to 1.5 and -0.30, respectively. The monomer subunit has a much higher affinity (P50 = 0.29 torr) and no cooperativity or Bohr effect.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of pH and temperature on the self-association of recombinant human interleukin-2 as studied by equilibrium sedimentation.

The self-association of recombinant human interleukin-2 (rhIL-2) in solution was investigated as a function of pH and temperature using equilibrium sedimentation. Studies were performed at pH 3.6, 6.5 and 8.2, at 1 degree C and 20 degrees C. A model assuming an ideal single molecular species describes the data observed at pH 6.5 at both temperatures. At pH 8.2, the data from both temperatures can be better described by a weak monomer-dimer association equilibrium. The values of the association constants obtained indicate the presence of less than 10% dimer at a concentration of 1 mg/ml at both temperatures. At pH 3.6, aggregates with a Z average molecular weight of over 35 times that of monomeric rhIL-2 were formed. The smallest associating species present under these conditions corresponds to the monomer, which produces aggregates with a wide range of molecular weights. The monomer appears to be in equilibrium with the smallest aggregates, in that a model describing an indefinite association fits the data obtained at the highest centrifugal speed. No model was found to successfully describe the association of the monomer into the much larger aggregates observed at lower speeds. This may be the result of the lack of rapid thermodynamic reversibility of the larger aggregates. Temperature was found to have no significant effect on the largest aggregates that were formed at pH 3.6.