Liquid-liquid phase separation in hemoglobins: distinct aggregation mechanisms of the beta6 mutants - PubMed (original) (raw)

Liquid-liquid phase separation in hemoglobins: distinct aggregation mechanisms of the beta6 mutants

Qiuying Chen et al. Biophys J. 2004 Mar.

Abstract

Reversible liquid-liquid (L-L) phase separation in the form of high concentration hemoglobin (Hb) solution droplets is favored in an equilibrium with a low-concentration Hb solution when induced by inositol-hexaphosphate in the presence of polyethylene glycol 4000 at pH 6.35 HEPES (50 mM). The L-L phase separation of Hb serves as a model to elucidate intermolecular interactions that may give rise to accelerated nucleation kinetics of liganded HbC (beta6 Lys) compared to HbS (beta6 Val) and HbA (beta6 Glu). Under conditions of low pH (pH 6.35) in the presence of inositol-hexaphosphate, COHb assumes an altered R-state. The phase lines for the three Hb variants in concentration and temperature coordinates indicate that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS and HbA. Over time, L-L phase separation gives rise to amorphous aggregation and subsequent formation of crystals of different kinetics and habits, unique to the individual Hb. The composite of R- and T-like solution aggregation behavior indicates that this is a conformationally driven event. These results indicate that specific contact sites, thermodynamics, and kinetics all play a role in L-L phase separation and differ for the beta6 mutant hemoglobins compared to HbA. In addition, the dense liquid droplet interface or aggregate interface noticeably participates in crystal nucleation.

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Figures

FIGURE 1

Formation of (A) COHbA, C, and S droplets and (B) droplet counts in the presence of IHP and PEG 4000 (50 mM HEPES at pH 6.35). The initial solution contained 1.0 g/dl COHb, 2:1 IHP:Hb, and 10% PEG 4000, 4°C. Bar = 20 _μ_m.

FIGURE 2

A classical concentration-temperature phase diagram. Modified from

www.poco.phy.cam.ac.uk/teaching/A\_Donald/Phase\_Diagrams.pdf

FIGURE 3

Binodal curves of COHbA, C, and S experimentally measured (solid line) and plotted from Eq. 1 (dashed line). (5% PEG 4000, 4:1 IHP:Hb, and 50 mM HEPES at pH 6.35.)

FIGURE 4

IHP and PEG modulation of binodal curves of L-L phase separations: COHbA, COHbC, and COHbS (50 mM HEPES at pH 6.35). (A) The IHP effect on COHbC and COHbA at 10% PEG, _T_L-L for COHbC >2.0 g/dl, is not attainable due to the formation of aggregates and crystals during measurement (see text). (B) The PEG effect on COHbC and COHbA at 4:1 IHP:COHb. (C) IHP and PEG significantly affect the binodal curve of COHbS. Under certain conditions (e.g., A and B), _T_L-L for COHbS is not attainable due to the formation of crystals and aggregates within the time of measurement (see text).

FIGURE 5

Chloride significantly affects the binodal curves of (A) COHbC and (B) COHbA (10% PEG, 4:1 IHP:Hb, and 50 mM HEPES at pH 6.35). Note that because COHbS (1.0–10.0 g/dl) forms needle crystals even in the presence of up to 0.1 M chloride, the chloride effect on the COHbS binodal curve is not attainable (see text).

FIGURE 6

Hemoglobin C crystals and aggregates after L-L phase separation. Crystals notably grow at the liquid droplet or aggregate interface edge (50 mM HEPES at pH 6.35, 1.0 g/dl COHbC, 4:1 IHP:Hb, and 10% PEG 4000, 20°C). (A) Less commonly, precrystalline structures of HbC form inside of liquid droplets by ∼2 h. (B, left image) A liquid droplet, and a denser liquid droplet with both tetragonal and orthorhombic crystals growing at the interface (∼2.5 to >5 h). (B, right image) A hexagonal crystal in the process of forming at the interface of a liquid droplet. (C) Crystals growing from the edge of aggregates by ∼5 h; crystals of different habits grow at the surface of a single aggregate (C, left image), or from different aggregates (C, middle and left images). (D) Hexagonal and tetragonal crystals form at ∼5 h. (D, left image) A liquid droplet, and a liquid droplet giving rise to a hexagonal crystal—the arrow points to the liquid droplet surface from which hemoglobin molecules appear to be recruited. (D, middle image) Hexagonal crystals formed from liquid droplets. (D, right image) Orthorhombic crystals formed from liquid droplets. It is known that crystals of different habits form under the same condition (e.g., McPherson, 1982) or within a single liquid droplet (Durbin and Feher, 1996). Color, at 1000× magnification; black and white, at 4000× magnification. (See Materials and Methods for explanation.)

FIGURE 7

The formation of COHbS crystals and aggregates in L-L phase separation (50 mM HEPES at pH 6.35, 1.0 g/dl COHbS, 4:1 IHP:Hb, and 10% PEG 4000, at 20°C). (A) Spherulite domains by ∼2 min; (B) needle crystals and bundles of needle crystals by ∼10 min; and (C) macrofibers and macroarrays by ∼10 min. At 4000× magnification. (See Materials and Methods for explanation.)

FIGURE 8

After L-L phase separation, COHbA forms orthorhombic crystals at higher Hb concentration and requires a significantly longer incubation time (>22 h). (50 mM HEPES at pH 6.35, 3.0 g/dl COHbA, 4:1 IHP:Hb, and 10% PEG 4000, at 20°C.)

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Liquid-liquid phase separation in hemoglobins: distinct aggregation mechanisms of the beta6 mutants - PubMed (original) (raw)