β7E–β132K salt bridge and sickle haemoglobin stability and conformation

Abstract

Summary. The liganded (R-state) form of sickle cell haemoglobin (HbS) is of particular relevance at non-polymerizing concentrations as oxy HbS exhibits unusual properties compared with oxy HbA: mechanical precipitability (resulting from surface denaturation), greater unfolding at an air-water interface and a tendency to oxidize more readily. In human haemoglobins, the b7 (A4) Glu residue forms an intrachain salt bridge with b132 (H10) Lys in both liganded and deoxy structures. In the present study, recombinant haemoglobins with substitutions in the b7 and b132 sites were studied in order to determine the role of the b7-b132 salt bridge on Hb conformational integrity and stability. The elimination of this interhelix bridge correlates with enhanced surface denaturation and conformational alterations in the central cavity 2,3-diphosphoglycerate (DPG) cleft and a1b2 interface. The A-helix b7 Ala substitution generates a class of conformational change at the DPG pocket and a1b2 interface that is distinct from that dictated by the H-helix b132 Ala substitution. These results are significant with regard to the communication pathway between the a1b1 and a1b2 interfaces, and the new understanding of Hb allostery dependent upon tertiary structural constraints caused by effector binding to the R-state.

Liganded Hemoglobin Structural Perturbations by the Allosteric Effector L35

“…Front-face optics is advantageous in that it permits a direct observation of HPT fluorescence in the presence of Hb (for a review, see Hirsch, 2000Hirsch, , 2003. Several central cavity-binding effectors, such as IHP and Cl ÿ , displace HPT from the central cavity of the altered R-state Hb as indicated by an intensity increase in HPT fluorescence (Hirsch et al, 1999;Chen et al, 2002;Fablet et al, 2003).…”

“…In the present study, we aim to clarify the various structural interpretations by spectroscopically exploring the binding properties of L35, a BZF derivative (Poyart et al, 1994), to the low pH altered R-state, correlated with site-specific structural changes in both heme and globin domains (Scott et al, 1985). The altered R-state has proven useful in solution studies probing central cavity differences in variant hemoglobins and in comparative effector binding studies (e.g., Hirsch et al, 1996Hirsch et al, , 1997Hirsch et al, , 1999Chen et al, 2002;Fablet et al, 2003). Using the low pH transition state, evidence is presented that shows L35 induces a transition species characterized by domain-specific tertiary and quaternary-like conformation within a global R-quaternary structure.…”