Thomas M. Laue

Iron and Hydrogen Peroxide Detoxification Properties of DNA-binding Protein from Starved Cells A FERRITIN-LIKE DNA-BINDING PROTEIN OF ESCHERICHIA COLI

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H2O2. Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H2O2 rather than by O2 as in ferritins. Two Fe2+ ions bind at each of the 12 putative dinuclear ferroxidase sites (PZ) in the protein according to the equation, 2Fe2+ + PZ→ [(Fe(II)2-P] FS Z + 2 + 2H+. The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)2-P] FS Z + 2 + H2O2 + H2O → [Fe(III)2O2(OH)-P] FS Z − 1 + 3H+, where two Fe(II) are oxidized per H2O2 reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of ∼500 Fe(III) according to the mineralization equation, 2Fe2+ + H2O2 + 2H2O → 2Fe(III)OOH(core) + 4H+, again with a 2 Fe(II)/H2O2 stoichiometry. The protein forms a similar ferric core with O2 as the oxidant, albeit at a slower rate. In the absence of H2O2 and O2, Dps forms a ferrous core of ∼400 Fe(II) by the reaction Fe2+ + H2O + Cl− → Fe(II)OHCl(core) + H+. The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H2O2. Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H2O2. These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H2O2. In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.

Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium

Analytical ultracentrifugation (AUC) is a versatile and powerful method for the quantitative analysis of macromolecules in solution. AUC has broad applications for the study of biomacromolecules in a wide range of solvents and over a wide range of solute concentrations. Three optical systems are available for the analytical ultracentrifuge (absorbance, interference, and fluorescence) that permit precise and selective observation of sedimentation in real time. In particular, the fluorescence system provides a new way to extend the scope of AUC to probe the behavior of biological molecules in complex mixtures and at high solute concentrations. In sedimentation velocity (SV), the movement of solutes in high centrifugal fields is interpreted using hydrodynamic theory to define the size, shape, and interactions of macromolecules. Sedimentation equilibrium (SE) is a thermodynamic method where equilibrium concentration gradients at lower centrifugal fields are analyzed to define molecule mass, assembly stoichiometry, association constants, and solution nonideality. Using specialized sample cells and modern analysis software, researchers can use SV to determine the homogeneity of a sample and define whether it undergoes concentration-dependent association reactions. Subsequently, more thorough model-dependent analysis of velocity and equilibrium experiments can provide a detailed picture of the nature of the species present in solution and their interactions.

The influence of charge distribution on self-association and viscosity behavior of monoclonal antibody solutions.

The present work investigates the influence of electrostatic surface potential distribution of monoclonal antibodies (MAbs) on intermolecular interactions and viscosity. Electrostatic models suggest MAb-1 has a less uniform surface charge distribution than MAb-2. The patches of positive and negative potential on MAb-1 are predicted to favor intermolecular attraction, even in the presence of a small net positive charge. Consistent with this expectation, MAb-1 exhibits a negative second virial coefficient (B₂₂), an increase in static structure factor, S((q→0)), and a decrease in hydrodynamic interaction parameter, H((q→0)), with increase in MAb-1 concentration. Conversely, MAb-2 did not show such heterogeneous charge distribution as MAb-1 and hence favors intermolecular repulsion (positive B₂₂), lower static structure factor, S((q→0)), and repulsion induced increase in momentum transfer, H((q→0)), to result in lower viscosity of MAb-2. Charge swap mutants of MAb-1, M-5 and M-7, showed a decrease in charge asymmetry and concomitantly a loss in self-associating behavior and lower viscosity than MAb-1. However, replacement of charge residues in the sequence of MAb-2, M-10, did not invoke charge distribution to the same extent as MAb-1 and hence exhibited a similar viscosity and self-association profile as MAb-2.

[19] Sedimentation equilibrium as thermodynamic tool

Publisher Summary This chapter introduces sedimentation equilibrium as thermodynamic tool. The chapter presents the short column method and diagnostic procedures for detecting the presence of (1) reversible macromolecular association, (2) sample heterogeneity, and (3) thermodynamic non-ideality. The quantitative analysis of equilibrium sedimentation is conducted. This chapter also focuses on the practical application of sedimentation equilibrium to thermodynamic analyses, with an emphasis on interacting systems. One of the virtues of sedimentation equilibrium is that it can be used to determine the molecular weight of both native and denatured molecules. Several methods are proposed for extracting thermodynamic information from sedimentation equilibrium data. The method preferred employs nonlinear least-squares analysis of the primary data, using the equations above to serve as the models. The principal advantages of this approach are that (1) the untransformed data are analyzed, thus minimizing distortion of the data points and the error on them; (2) the fitting functions are derived from thermodynamic first principles, thus lending confidence to the values obtained from the analysis; and (3) an excellent computer program, NONLIN, has been refined for use with sedimentation equilibrium data.

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions: Part 1. Unfolding, Colloidal Interactions, and Formation of High-Molecular-Weight Aggregates

Monomeric and aggregated states of an IgG1 antibody were characterized under acidic conditions as a function of solution pH (3.5-5.5). A combination of intrinsic/extrinsic fluorescence (FL), circular dichroism, calorimetry, chromatography, capillary electrophoresis, and laser light scattering were used to characterize unfolding, refolding, native colloidal interactions, aggregate structure and morphology, and aggregate dissociation. Lower pH led to larger net repulsive colloidal interactions, decreased thermal stability of Fc and Fab regions, and increased solubility of thermally accelerated aggregates. Unfolding of the Fab domains, and possibly the CH3 domain, was inferred as a key step in the formation of aggregation-prone monomers. High-molecular-weight soluble aggregates displayed nonnative secondary structure, had a semi-rigid chain morphology, and bound thioflavin T (ThT), consistent with at least a portion of the monomer forming amyloid-like structures. Soluble aggregates also formed during monomer refolding under conditions moving from high to low denaturant concentrations. Both thermally and chemically induced aggregates showed similar ThT binding and secondary structural changes, and were noncovalent based on dissociation in concentrated guanidine hydrochloride solutions. Changes in intrinsic FL during chemical versus thermal unfolding suggest a greater degree of structural change during chemical unfolding, although aggregation proceeded through partially unfolded monomers in both cases.

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions.

Specific‐ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister‐ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen‐egg white lysozyme (HEWL) as a direct and differential measure of ion‐association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li+, Na+, K+, Rb+, Cs+, GdnH+, and Ca2+), identity. The Q* decreased in the order F− > Cl− > Br− > NO  3− ∼ I− > SCN− > ClO  4− ≫ SO  42− , demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO  42− anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.

NUTS and BOLTS: applications of fluorescence-detected sedimentation.

Analytical ultracentrifugation is a widely used method for characterizing the solution behavior of macromolecules. However, the two commonly used detectors, absorbance and interference, impose some fundamental restrictions on the concentrations and complexity of the solutions that can be analyzed. The recent addition of a fluorescence detector for the XL-I analytical ultracentrifuge (AU-FDS) enables two different types of sedimentation experiments. First, the AU-FDS can detect picomolar concentrations of labeled solutes, allowing the characterization of very dilute solutions of macromolecules, applications we call normal use tracer sedimentation (NUTS). The great sensitivity of NUTS analysis allows the characterization of small quantities of materials and high-affinity interactions. Second, the AU-FDS allows characterization of trace quantities of labeled molecules in solutions containing high concentrations and complex mixtures of unlabeled molecules, applications we call biological on-line tracer sedimentation (BOLTS). The discrimination of BOLTS enables the size distribution of a labeled macromolecule to be determined in biological milieus such as cell lysates and serum. Examples that embody features of both NUTS and BOLTS applications are presented along with our observations on these applications.

Altered affinity of CBFβ-SMMHC for Runx1 explains its role in leukemogenesis

Chromosomal translocations involving the human CBFB gene, which codes for the non-DNA binding subunit of CBF (CBFβ), are associated with a large percentage of human leukemias. The translocation inv(16) that disrupts the CBFB gene produces a chimeric protein composed of the heterodimerization domain of CBFβ fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (CBFβ-SMMHC). Isothermal titration calorimetry results show that this fusion protein binds the Runt domain from Runx1 (CBFα) with higher affinity than the native CBFβ protein. NMR studies identify interactions in the CBFβ portion of the molecule, as well as the SMMHC coiled-coil domain. This higher affinity provides an explanation for the dominant negative phenotype associated with a knock-in of the CBFB-MYH11 gene and also helps to provide a rationale for the leukemia-associated dysregulation of hematopoietic development that this protein causes.

Biophysical characterization of interactions between the core binding factor α and β subunits and DNA

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFα subunit and a non‐DNA binding CBFβ subunit that increases the affinity of CBFα for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFα:β:DNA complex. Dissociation constants for all four equilibria involving the CBFα Runt domain, CBFβ, and DNA were defined. Conformational changes associated with interactions between CBFα, CBFβ, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFβ ‘locks in’ a high affinity DNA binding conformation of the CBFα Runt domain.

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal DIRECT ANALYSIS BY FLUORESCENCE-DETECTED ANALYTICAL ULTRACENTRIFUGATION

Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys10) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescence-detected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.