Characterization of an associated equilibrium folding intermediate of bovine growth hormone

In the preceding paper [Havel, H. A., Kauffman, E. W., Plaisted, S. M., & Brems, D. N. (1986) Biochemistry (preceding paper in this issue)], an associated intermediate was shown to be highly populated during the equilibrium denaturation of bovine growth hormone. In this paper, we describe its partial characterization and propose a mechanism for association. The associated equilibrium intermediate is populated under conditions that induce partial denaturation and at protein concentrations greater than 0.2 mg/mL. The remaining nativelike helical structure present in the partially denatured species is implicated in the mechanism of association as demonstrated by similar concentration dependencies and thermal stabilities of the helix and the associated equilibrium intermediate. Furthermore, it is suggested that a putative amphiphilic helix from residues 110-127 plays a critical role in the association as demonstrated by a diminution of the associated equilibrium intermediate when mixed with the peptide fragment 96-133. A model is proposed to account for these results in which partial denaturation exposes the segment of the protein corresponding to the hydrophobic face of the putative amphiphilic helix 110-127. This metastable form is the species from which association occurs. Association is stabilized by the hydrophobic interactions resulting from intermolecular packing of the lipophilic faces of the helices. The implications of these results to protein folding studies are described.     

Terrigenous sediment-dominated reef platform infilling: an unexpected precursor to reef island formation and a test of the reef platform size–island age model in the Pacific

Low-lying coral reef islands are considered highly vulnerable to climate change, necessitating an improved understanding of when and why they form, and how the timing of formation varies within and among regions. Several testable models have been proposed that explain inter-regional variability as a function of sea-level history and, more recently, a reef platform size model has been proposed from the Maldives (central Indian Ocean) to explain intra-regional (intra-atoll) variability. Here we present chronostratigraphic data from Pipon Island, northern Great Barrier Reef (GBR), enabling us to test the applicability of existing regional island evolution models, and the platform size control hypothesis in a Pacific context. We show that reef platform infilling occurred rapidly (~4–5 mm yr⁻¹) under a “bucket-fill” type scenario. Unusually, this infilling was dominated by terrigenous sedimentation, with platform filling and subsequent reef flat formation complete by ~5000 calibrated years BP (cal BP). Reef flat exposure as sea levels slowly fell post highstand facilitated a shift towards intertidal and subaerial-dominated sedimentation. Our data suggest, however, a lag of ~1500 yr before island initiation (at ~3200 cal BP), i.e. later than that reported from smaller and more evolutionarily mature reef platforms in the region. Our data thus support: (1) the hypothesis that platform size acts to influence the timing of platform filling and subsequent island development at intra-regional scales; and (2) the hypothesis that the low wooded islands of the northern GBR conform to a model of island formation above an elevated reef flat under falling sea levels.

     

Assignment of 1H, 13C and 15N resonances of the reduced human IgG1 CH3 domain

Here we report the NMR resonance assignments for the reduced form of human IgG1 C_H3 domain, a 26 kDa dimer in solution (residues 341–447). The assignments have been deposited in the BioMagResBank with a BMRB accession number of 15204.     

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.     

pH Dependence of structural stability of interleukin-2 and granulocyte colony-stimulating factor

After a cytokine binds to its receptor on the cell surface (pH approximately 7), the complex is internalized into acidic endosomal compartments (pH approximately 5-6), where partially unfolded intermediates can form. The nature of these structural transitions was studied for wild-type interleukin-2 (IL-2) and wild-type granulocyte colony-stimulating factor (G-CSF). A noncoincidence of denaturation transitions in the secondary and tertiary structure of IL-2 and tertiary structural perturbations in G-CSF suggest the presence of an intermediate state for each, a common feature of this structural family of four-helical bundle proteins. Unexpectedly, both IL-2 and G-CSF display monotonic increases in stability as the pH is decreased from 7 to 4. We hypothesize that such cytokines with cell-based clearance mechanisms in vivo may have evolved to help stabilize endosomal complexes for sorting to lysosomal degradation. We show that mutants of both IL-2 and G-CSF have differential stabilities to their wild-type counterparts as a function of pH, and that these differences may explain the differences in ligand trafficking and depletion. Further understanding of the structural changes accompanying unfolding may help guide cytokine design with respect to ligand binding, endocytic trafficking, and, consequently, therapeutic efficacy.     

Second polar body inclusion results in diploid/triploid mixoploidy

We report three cases with a typical diploid/triploid mixoploidy. Cytogenetic analysis showed a normal diploid karyotype in peripheral blood lymphocytes and a mixture of diploid and triploid cells in skin fibroblasts. We analysed microsatellite markers in patients blood lymphocytes and skin fibroblasts and compared the results with the microsatellite markers in the parents. The extra haploid set was in all three cases of maternal origin. In one case the markers were not very informative but in two cases pericentromeric markers showed a single dose of one paternal allele and a double dose of one maternal allele, more telomeric markers showed one paternal allele and two different maternal alleles. These observations can only be explained by the inclusion of the second polar body in one of the blastomeres at the cleavage stage.     

Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation

The concentration-dependence of the diffusion and sedimentation coefficients (k_D and k_s, respectively) of a protein can be used to determine the second virial coefficient (B₂), a parameter valuable in predicting protein-protein interactions. Accurate measurement of B₂ under physiologically and pharmaceutically relevant conditions, however, requires independent measurement of k_D and k_s via orthogonal techniques. We demonstrate this by utilizing sedimentation velocity (SV) and dynamic light scattering (DLS) to analyze solutions of hen-egg white lysozyme (HEWL) and a monoclonal antibody (mAb1) in different salt solutions. The accuracy of the SV-DLS method was established by comparing measured and literature B₂ values for HEWL. In contrast to the assumptions necessary for determining k_D and k_s via SV alone, k_D and ks were of comparable magnitudes, and solution conditions were noted for both HEWL and mAb1 under which 1), k_D and k_s assumed opposite signs; and 2), k_D ≥ k_s. Further, we demonstrate the utility of k_D and k_s as qualitative predictors of protein aggregation through agitation and accelerated stability studies. Aggregation of mAb1 correlated well with B₂, k_D, and k_s, thus establishing the potential for k_D to serve as a high-throughput predictor of protein aggregation.