Crystallization mechanisms of hemoglobin C in the R state.

Crystallization of the mutated hemoglobin, HbC, which occurs inside red blood cells of patients expressing betaC-globin and exhibiting the homozygous CC and the heterozygous SC (in which two mutant beta-globins, S and C, are expressed) diseases, is a convenient model for processes underlying numerous condensation diseases. As a first step, we investigated the molecular-level mechanisms of crystallization of this protein from high-concentration phosphate buffer in its stable carbomonoxy form using high-resolution atomic force microscopy. We found that in conditions of equilibrium with the solution, the crystals' surface reconstructs into four-molecule-wide strands along the crystallographic a (or b) axis. However, the crystals do not grow by the alignment of such preformed strands. We found that the crystals grow by the attachment of single molecules to suitable sites on the surface. These sites are located along the edges of new layers generated by two-dimensional nucleation or by screw dislocations. During growth, the steps propagate with random velocities, with the mean being an increasing function of the crystallization driving force. These results show that the crystallization mechanisms of HbC are similar to those found for other proteins. Therefore, strategies developed to control protein crystallization in vitro may be applicable to pathology-related crystallization systems.

Flexibility and nucleation in sickle hemoglobin.

We have studied the self-assembly of Hemoglobin C-Harlem (HbC-Harlem), a double mutant of hemoglobin that possesses the beta6 Glu-->Val mutation of sickle hemoglobin (HbS) plus beta73 Asp-->Asn. By electron microscopy we find it forms crystals, rather than the wrapped multistranded fibers seen in HbS. Fourier transforms of the crystals yield unit cell parameters indistinguishable from crystals of HbS. Differential interference contrast (DIC) microscopy and birefringence also show crystal formation rather than the polymers or domains seen for HbS, while the growth patterns showed radiating crystal structures rather than simple linear crystalline forms. The solubility of the assembly was measured using a photolytic micromethod over a temperature range of 17-31 degrees C in 0.15 M phosphate buffer and found to be essentially the same as that of fibers of HbS. The assembly kinetics were observed by photolysis of the carbon monoxide derivative, and the mass of assembled hemoglobin was found to grow exponentially, with onset times that were stochastically distributed for small volumes. The stochastic onset of assembly showed strong concentration dependence, similar to but slightly greater than that seen in sickle hemoglobin nucleation. These observations suggest that like HbS, HbC-Harlem assembly proceeds by a homogeneous nucleation process, followed by heterogeneous nucleation. However, relative to HbS, both homogeneous and heterogeneous nucleation are suppressed by almost 11 orders of magnitude. The slowness of nucleation can be reconciled with the similarity of the solubility to HbS by an increase in contact energy coupled with a decrease in vibrational entropy recovered on assembly. This also explains the linearity of the double-strands, and agrees with the chemical nature of the structural replacement.

Differential pathways in oxy and deoxy HbC aggregation/crystallization.

CC individuals, homozygous for the expression of beta(C)-globin, and SC individuals expressing both beta(S) and beta(C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype. To date, the question remains as to why HbC forms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal formation induced under conditions of concentrated phosphate buffer (2g% Hb, 1. 8M potassium phosphate buffer) and viewed by differential interference contrast microscopy. Oxy HbC formed isotropic amorphous aggregates with subsequent tetragonal crystal formation. Also observed, but less numerous, were twisted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in the oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored the formation of a greater morphologic variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed flat, hexagonal crystals. These results suggest: (1) the Lys substitution at beta6 evokes a crystallization process dependent upon ligand state conformation [i. e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) the oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of HbC that energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.