WAXS studies of the structural diversity of hemoglobin in solution

Abstract

Specific ligation states of hemoglobin are, when crystallized, capable of taking on multiple quaternary structures. The relationship between these structures, captured in crystal lattices, and hemoglobin structure in solution remains uncertain. Wide-angle X-ray solution scattering (WAXS) is a sensitive probe of protein structure in solution that can distinguish among similar structures and has the potential to contribute to these issues. We used WAXS to assess the relationships among the structures of human and bovine hemoglobins in different liganded forms in solution. WAXS data readily distinguished among the various forms of hemoglobins. WAXS patterns confirm some of the relationships among hemoglobin structures that have been defined through crystallography and NMR and extend others. For instance, methemoglobin A in solution is, as expected, nearly indistinguishable from HbCO A. Interestingly, for bovine hemoglobin, the differences between deoxy-Hb, methemoglobin and HbCO are smaller than the corresponding differences in human hemoglobin. WAXS data were also used to assess the spatial extent of structural fluctuations of various hemoglobins in solution. Dynamics has been implicated in allosteric control of hemoglobin, and increased dynamics has been associated with lowered oxygen affinity. Consistent with that notion, WAXS patterns indicate that deoxy-Hb A exhibits substantially larger structural fluctuations than HbCO A. Comparisons between the observed WAXS patterns and those predicted on the basis of atomic coordinate sets suggest that the structures of Hb in different liganded forms exhibit clear differences from known crystal structures.

Figure 1

Comparisons of WAXS patterns from HbCO A with different hemoglobins collected from samples at 50 mg/ml at 4°C (a) Comparison with the pattern from deoxy-Hb A. (b) met-Hb A. (c) rHbCO A (αV96W/βN108K), (d) di-α rHbCO. Differences among patterns are also displayed including a comparison of the changes in intensity going from HbCO A to deoxyHb A with the changes in going from (e) HbCO A to r HbCO (αV96W/βN108K) and (f) HbCOA to di-α-rHbCO.

Figure 2

Comparison of WAXS patterns from human and bovine hemoglobins collected from samples at 50 mg/ml at 4°C. (a) Comparison of patterns from bovine metHb, HbCO and deoxyHb. (b) Bovine metHb with human metHb A. (c) Bovine deoxyHb with human deoxyHb A. (d) A comparison of the differences between WAXS patterns from metHb and deoxyHb for human and bovine hemoglobins.

Figure 3

Computed and observed WAXS patterns from Hb. (a) Comparison of computed WAXS patterns from HbCO A (black) and deoxy-Hb (red). (b) Comparison of a computed WAXS pattern with that observed from HbCO A at 50 mg/ml and 4°C. (c) Comparison of a computed WAXS pattern with that observed from myoglobin.

Figure 4

Dependence of WAXS patterns on protein concentration. Patterns from samples at 10, 20 and 50 mg/ml are shown for (a) HbCO A; (b) deoxy-Hb A; and (c) bovine deoxy HbA.

Figure 5

Differences between WAXS patterns at 20 mg/ml and 50 mg/ml. Each plot contains the differences from HbCO A (black) compared to that of another Hb (red). (a) di-α-rHbCO. (b) deoxy-Hb A divided by 2. The differences are so large for deoxy-Hb (see Figure 5), that they were divided by 2 in this figure in order to aid visual comparisons. (c) human met-Hb A. (e) rHbCO (αV96W/βN108K).