Inhibition of Protein Aggregation: Supramolecular Assemblies of Arginine Hold the Key

BACKGROUND: Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. METHODOLOGY: We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimer's amyloid beta 1-42 (Abeta(1-42)) peptide is modified. Changes in the hydrodynamic volume of Abeta(1-42) in the presence of arginine measured by size exclusion chromatography show that arginine binds to Abeta(1-42). Arginine increases the solubility of Abeta(1-42) peptide in aqueous medium. It decreases the aggregation of Abeta(1-42) as observed by atomic force microscopy. CONCLUSIONS: Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation

Hyperthermophile Protein Behavior: Partially-Structured Conformations of <i>Pyrococcus furiosus</i> Rubredoxin Monomers Generated through Forced Cold-Denaturation and Refolding

<div><p>Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of <i>Pyrococcus furiosus</i> triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from <i>Pyrococcus furiosus</i> (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as ‘<i>Trishanku</i>’ unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native ‘<i>Trishanku</i>’ refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.</p></div

TUI data.

<p>Spectral changes in PfRd in the presence of 6°C/min. Parameters describing individual treatments and temperatures applicable to each curve are mentioned alongside as inset within the relevant panels. <i>Panel A</i> : Changes in PfRd's mean residue ellipticity CD signal at 222 nm as a function of heating and cooling, monitored during two successive rounds of heating and cooling of the same sample. <i>Panel B</i> : Far-UV CD spectra of PfRd collected at different temperatures, before heating, after heating, and after cooling, through two successive rounds of heating and cooling, corresponding to Panel A. <i>Panel C</i> : Fluorescence emission spectra of PfRd collected at 25°C, before heating, and after the first, and second, rounds of heating and cooling.</p

Figure 9

<p>A ribbon-diagram structural representation of holo-PfRd showing the bound iron atom (orange sphere), as well as five surface lysine residue (blue sidechains; K2, K6, K28, K45, K50), seven surface aspartate residues (green sidechains; D13, D15, D18, D20, D34, D35, D53) and six surface glutamate residues (red sidechains : E14, E30, E31, E47, E49, E52).</p

Cold denaturation behaviour of PfRd; its reversibility and the effect of B-ME.

<p><i>Panel A</i>: Cold denaturation in PfRd was inhibited by B-ME added at 25°C before initial heating (black curve, 25°C). <i>Panel B</i> :Reversal of cold denaturation, observed through monitoring of reversal in far-UV CD spectral features (secondary structural content). Black curve shows the spectrum of the native form in the presence of Gdm.HCl and B-ME prior to heating; the red curve shows the spectrum at 98°C after first heating; the green curve shows the spectrum at 25°C after cooling from 98°C.<i>Panel C</i> : Cold denaturation in PfRd reversed by B-ME added at 98°C after first heating (black curve, 98°C). No further cold-denaturation (blue curve) seen; instead cold-denaturation is reversed. <i>Panel D</i> : Cold denaturation in PfRd reversed by B-ME added at 25°C. The first heating-cooling cycle is shown by the black (heating) and red (cooling) curves, showing cold-denaturation. Then B-ME was added. The second heating cycle is shown by the green (heating) and blue (cooling) curves, and B-ME is seen to have abrogated cold-denaturation and returned the protein to original state. <i>Panel E</i> :In contrast to the curves in panel D, where B-ME was added at 25°C, here B-ME was added at 98°C, after the second heating. Again, only after B-ME is added, is the reversal of cold-denatuation seen. <i>Panel F</i> : Cold denaturation in PfRd can be reversed significantly by B-ME when added after the first heating-cooling cycle, but only if the temperature at which it is added is above 65–70°C. The black curve is the spectrum of native PfRd and the red curve is the spectrum of cold-denatured PfRd. All other curves represent PfRd's spectrum after heating to a certain temperature (mentioned in the panel), adding B-ME, and cooling back to room temperature. It is seen that if B-ME is added at temperatures below 70°C, the spectrum does not return to that of native PfRd. Otherwise, the spectrum returns to that of PfRd.</p

TUI data.

<p>Spectral changes in PfRd as a function of heating (H) and cooling (C) in the presence of denaturant. Individual values of temperatures, Gdm.HCl concentrations, rates of heating, and manners of treatment applicable to each panel/curve are mentioned as insets within panels. H+C indicates that the sample has been heated from 25°C to 103°C, and cooled back to 25°C. <i>Panel A</i> : Control far-UV CD spectra of PfRd in the absence of Gdm.HCl, before, after heating, and after cooling. <i>Panel B</i> : Far-UV CD spectra of PfRd collected at 25°C, before heating, and after heating and cooling using different treatments, as mentioned. <i>Panel C</i> : Fluorescence emission spectra of PfRd collected at 25°C, before heating, and after heating and cooling using different treatments, as mentioned, corresponding to samples for which CD spectra are shown in Panel B. <i>Panel D</i> : UV-visible absorption spectra of PfRd collected at 25°C, before heating, and after heating and cooling, using different treatments as mentioned, corresponding to some of the samples shown in Panels B and C, and a representative spectrum for a sample using a lower Gdm.HCl concentration.</p

Heating and cooling between 25°C and either 86°C (<i>Panel</i> A) or 100°C (<i>Panel B</i>), in the presence of B-ME and Gdm.HCl.

<p>The negative band at 315-bound iron while other bands mainly represent PfRd-bound iron in the oxidized state. The higher temperature can be seen to provide more access to B-ME to reduce PfRd-bound iron.</p

TRI data.

<p>Comparison of temperature-dependent changes in the far-UV CD spectra of TRI-I (<i>Panel A</i>) and TRI-II (<i>Panel B</i>). The values of the temperatures at which data was collected are mentioned within the panels as inset.</p

TRI data.

<p>Comparison of the characteristics of native PfRd (control) with the characteristics of two non-native PfRd states, TRI-I and TRI-II, obtained through dialysis-based removal of Gdm.HCl from PfRd samples, following (for the TRI-I sample) heating 1°C/min to 103°C at and cooling to 25°C at the same rate, and separately (for the TRI-II sample) incubation at 103°C for 1 hour, followed by cooling to 25°C. <i>Panel A</i> : Gel filtration chromatograms on Bio-Sil SEC-250 columns. <i>Panel B</i> : Native (17%) PAGE of native PfRd (lane 1), TRI-I (lane 2), TRI-II (lane 3); subtilisin-treated samples of native PfRd (lane 4), TRI-I (lane 5), TRI-II (lane 6); and trypsin-treated samples of native PfRd (lane 7), TRI-I (lane 8), TRI-II (lane 9). <i>Panel C</i> : Fluorescence emission spectra of native (control) and TRI-I and TRI-II states of PfRd. <i>Panel D</i> : Far-UV CD spectra of native (control) and TRI-I and TRI-II states of PfRd.</p