Net Interactions among Native and Denatured Lysozyme Monomers.

<p>(<b>A</b>) Debye plot of the static light scattering intensity (KC/R) <i>vs.</i> lysozyme concentration C at T = 20°C. The positive slope of these curves indicates that the interactions among the lysozyme monomers are predominately repulsive. This repulsion becomes screened out once NaCl concentrations reach about 400 mM. (<b>B</b>) Plot of the static interaction parameter k_s (which is proportional to the slope of KCp/R <i>vs.</i> Cp) <i>vs.</i> salt concentration for the Data in A. The dotted line is a guide to the eye indicating how repulsion decreases with increasing salt concentration. The two dashed vertical lines mark the switch of lysozyme aggregation from monomeric (MF) to oligomeric fibril (OF) assembly and, eventually, precipitate formation (P). (<b>C</b>) Change in net interactions as lysozyme monomers undergo thermal denaturation in the presence of 50 mM (○) and 200 mM (▪) NaCl. The vertical dashed line indicates the onset of thermal denaturation at 50°C. Note that, the prevailing intermolecular interactions remain repulsive (positive K_s values) even after thermal denaturation. At the same time, denaturation at 50 mM NaCl makes lysozyme slightly more repulsive while the monomers become less repulsive following denaturation at 200 mM NaCl.</p

Monomeric vs. Oligomeric Assembly Pathways for Lysozyme Amyloid Fibrils.

<p>(<b>A</b>) <i>In situ</i> particle size distributions at different stages of growth and corresponding temporal evolution of the detected aggregate peaks during lysozyme fibril growth at 50 mM NaCl (left two panels) <i>vs.</i> 175 mM NaCl (right two panels), as obtained from dynamic light scattering measurements. The temporal evolution of the aggregate peak radii (1A panel 3&4) highlights the dramatic difference in lag periods (see vertical dashed line) and distinctly different nucleation signatures: Low-salt samples always yielded two peaks while only a single peak nucleated at elevated salt concentrations (<b>B</b>) Morphology of growth intermediates in the presence of 50 mM NaCl (top row) <i>vs.</i> 175 mM NaCl (bottom row), as observed with atomic force microscopy. The vertical dashed line separates samples taken before and after the nucleation event detected by DLS. The false color scale indicates the height of the different aggregates in nm. The scale bars represent 50 nm, except for the 200 nm scale bars in the last image in either series. AFM images and aggregate dimensions for the 175 mM data are adapted from our earlier work (Hill et al, 2009). They are representative of the behavior observed throughout the "intermediate" salt concentrations (150 mM to 350 mM) associated with the oligomeric assembly regime. (<b>C</b>) Cross sectional areas for the various aggregates in (B) measured with calibrated AFM tips. Note the distinctly different cross sections for aggregates along the two different assembly pathways. <i><u>Top</u></i>: Cross-sectional areas of monomers, monomeric filaments and mature lysozyme fibrils grown at 50 mM NaCl. At low salt, no globular oligomeric species are detected. The cross sections for monomers and monomeric filaments are identical then increase by a factor of three for mature fibrils. <i><u>Bottom</u></i>: At intermediate salt concentrations, ellipsoidal oligomers are formed well before the nucleation event seen in DLS. These oligomers have a volume close to eight monomers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018171#pone-0018171-t001" target="_blank">Table 1</a>). The filaments emerging after nucleation have a cross section identical to that of the ellipsoidal oligomers. Late stage mature fibrils, in turn, had cross sectional areas close to two oligomeric filaments.</p

Effects of Salt-mediated Charge Screening on Denatured Monomers.

<p>The schematic indicates how the spatial extent (Debye screening length λ_D) of salt-mediated charge screening changes the character of the net interactions among denatured monomers and favors the formation of different aggregate geometries. The black curvy line represents the protein backbone while the blue perimeter symbolizes the short-range attractive protein interactions (hydrophobic, dipole-dipole, hydrogen bonding). Individual charged residues are represented by small positive spheres, and the extent of charge screening mediated by the salt ions is indicated as a red cloud surrounding the charge residues. At low salt concentrations, (monomeric assembly pathway) individual charges on the same monomer strongly repel each other and those on neighboring monomers. Only those few conformations of denatured monomers that can form intermolecular bonds similar to those in the native monomer are aggregation competent. In addition, charge repulsion among monomers will favor extended, polymeric structures for intermediates since that will separate the monomer charges from each other while preserving sufficient intermolecular contacts. When salt screening reduces λ_D below the separation of charged residues (oligomeric assembly pathway) along the monomer backbone, charge repulsion within a given monomer and, concurrently, among several aggregated monomers is significantly reduced. This favors the formation of more compact (oligomeric) aggregate assemblies and requires fewer monomers to share their hydrophobic cores to overcome the residual charge repulsion and loss in configurational entropy. Finally, when λ_D becomes comparable in range to the attractive interactions, the charge restrictions on "suitable" aggregate morphologies and favorable monomer conformation fall by the wayside and the denatured monomers precipitate randomly out of solution.</p

Precipitate Formation of Amyloidogenic Lysozyme.

<p>(<b>A</b>) AFM image of precipitates and their corresponding height distributions observed shortly after the onset of aggregation. (<b>B</b>) DLS aggregate peaks of lysozyme in 400 mM NaCl before and right after partial denaturation of lysozyme (see vertical dashed line). (<b>C</b>) Congo Red spectra of native lysozyme (—) and lysozyme precipitates (▪) are indistinguishable. In contrast, mature fibrils grown at lower salt concentrations (open circles) induce the red shift and shoulder characteristic for binding to amyloid fibrils.</p

Comparison of metal ion binding sites for four OLFs and biophysical analysis for npoh-OLF and glio-OLF.

<p>(a) Metal binding sites in npoh-OLF. (b) Metal binding sites in myoc-OLF (PDB code 4WXU). (c) Metal binding sites in lat3-OLF (PDB code 5AFB). (d) Metal binding site in <i>M</i>. <i>musculus</i> glio-OLF. Lower panels show interacting distances ≤ 2.7 Å. For (a), (d), 2F_o-F_c electron density is contoured at 1σ. (e) Quin-2 fluorescence (in a.f.u., arbitrary fluorescence units) due to Ca²⁺ binding under native and denaturing conditions. Denaturing concentrations were 1.4 M GdHCl for npoh-OLF, and 5 M GdHCl for glio-OLF. (f) Chemical unfolding curves of npoh-OLF and glio-OLF monitored by the change in maximum intrinsic tryptophan fluorescence using GdHCl (left) and urea (right). Concentration at unfolding midpoint indicated in parentheses.</p

Structural features of npoh-OLF (yellow) and glio-OLF (purple).

<p>(a) Overlay of npoh-OLF and glio-OLF in two orientations with strands and blades labeled (r.m.s.d. over Cα atoms is 1.456 Å). (b) Disulfide bond in npoh-OLF (left) and corresponding cation-π interaction in glio-OLF (right). (c) Overview of molecular clasp region highlighting Pro, Tyr residues discussed in text; polar contacts < 3.5 Å are depicted as black dashes (left). Crystal contact of npoh-OLF involving residues from the molecular clasp and an outer strand of blade A from an adjacent symmetry-related molecule (right).</p

Comparison of top surface features, loop B-10/C-11, and related cation-π interaction.

<p>Comparison of (a) myoc-OLF (PDB code 4WXU), (b) npoh-OLF, and (c) <i>M</i>. <i>musculus</i> glio-OLF. Left: surface representation of loop region; right: key side chain interactions presented as sticks in two orientations. (d) Overlay of Lys/Tyr cation-π interaction conserved for myoc-OLF (blue-green), npoh-OLF (yellow), and lat3-OLF (orange) but disrupted for glio-OLF (purple).</p

Analysis of Thermal Stabilization of OLF domains.

<p>^aT_m measured in 10 mM Hepes, 200 mM NaCl pH 7.5 (Buffer A) with or without 0.75 mg/mL of GAG. For myoc-OLF, T_m is ~53°C in Buffer A [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130888#pone.0130888.ref061" target="_blank">61</a>].</p><p>Analysis of Thermal Stabilization of OLF domains.</p

Electrostatic surface representations and biochemical analysis of nucleotide and heparin binding for npoh-OLF.

<p>(a) Electrostatic surfaces of npoh-OLF and glio-OLF at top and bottom faces. (b) Electrostatic surfaces of myoc-OLF (PDB code 4WXU) and lat3-OLF (PDB code 5AFB). Surface potentials are colored negative (red, -5 kT/e^-) to positive (blue, + 5 kT/e^-). (c) Extraction analysis reveals small nucleotide stretches bound to npoh-OLF. (d) Low affinity binding of npoh-OLF to heparin column; no binding occurs with buffers at physiological ionic strength. (e) Commercial antibodies, anti-npoh-OLF and anti-myocilin (H130), lack specificity and detect npoh-OLF, myoc-OLF, and glio-OLF compared to custom myocilin antibody prepared in this study.</p

Thermodynamic parameters for metals binding to DHNTPase.

<p>Thermodynamic parameters for metals binding to DHNTPase.</p