Conformational Changes in Hemoglobin S (βE6V) Imposed by Mutation of the βGlu7–βLys132 Salt Bridge and Detected by UV Resonance Raman Spectroscopy

Abstract

The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, ␤Glu 7 -␤Lys 132 , are dependent on which residue of the pair is modified. The ␤K132A mutants exhibit the spectroscopic signatures of the R 3 T state transition in both the "hinge" and "switch" regions of the ␣ 1 ␤ 2 interface. Both singly and doubly mutated hemoglobin (Hb) ␤⌭7〈 exhibit the switch region signature for the R 3 T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb ␤⌭7〈 mutants. The observed large decrease in the W3 ␣14␤15 band intensity for doubly mutated Hb ␤⌭7〈 is attributed to an enhanced separation in the A helix-E helix tertiary contact of the ␤ subunits. The results for the Hb A ␤Glu 7 -␤Lys 132 salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the ␣ 1 ␤ 2 dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.Sickle cell hemoglobin (Hb S, 1 ␤6(A3) Glu 3 Val) exhibits the property of anomalous and pathologic self-assembly. DeoxyHb S forms polymers in the erythrocyte, which leads to microvascular blockage, organ damage, and often premature death. Structure-based drug design requires knowledge of optimal polymer disruption sites. The specific interaction in the Hb S polymer involves the steric fit of the mutated hydrophobic ␤6 donor site in a hydrophobic acceptor site located in an adjacent Hb S tetramer. It is well known that the hydrophobicity and the stereospecificity of the donor site are essential to the initiation of the polymerization.Lesecq et al.(1, 2) have been investigating whether modification of the polarity close to the ␤6 site could influence the packing of the donor and acceptor sites, thus modifying the polymerization process. Replacing the hydrophilic Glu ␤7(A4) residue with a hydrophobic Ala residue resulted in a decreased polymerization of the doubly mutated rHb ␤E6V/E7A. It was postulated (1, 2) that the loss of the normal salt bridge between ␤Glu 7 (A4) and ␤Lys 132 (H10) in the rHb ␤E7A mutants might lead to an alteration in both the position and the mobility of the A helix, illustrated in Fig. 1. These alterations of the A helix might result in a misfit between the donor and acceptor sites, which could explain the observed diminution in polymerization. It follows from this hypothesis that modifying the other partner of the salt bridge, ␤Lys 132 (H10), should have similar consequences on polymerization (2).Visible resonance Raman spectroscopy is very useful in providing detailed information relating to the influence of tertiary and quaternary structure upon specific heme-related vibrational degr...

β7E–β132K salt bridge and sickle haemoglobin stability and conformation

“…In other words, the class of conformational change within the DPG pocket, as a result of the loss of this bridge, is dictated by the unique substitution specific to the A‐ or H‐helix. These findings agree with UVRR studies demonstrating a distinct conformation induced not only by the altered salt bridge, but also by the specific substituted residue in the bridge ‘partnership’ (Juszczak et al, 2003). Therefore, it is further demonstrated here that the β7–β132 salt bridge participates in maintaining the integrity of the DPG binding site.…”

“…As previously noted, significant differences in the position of the A–E helix are reported for the β6 mutants (Hirsch et al , 1996, 1999) and, in a separate study using UVRR, a further weakening of the hydrogen bond between Trp β15 (A‐helix) and Ser β72 (E‐helix) was reported for the double mutant E6V/E7A (Juszczak et al, 2003). The consequence of this weakened bond would be an enhanced separation of the A‐helix away from the E‐helix which supports the earlier postulate that the loss of the normal salt bridge between β7 Glu (A4)–β132 Lys(H10) might lead to an alteration in both the position and mobility of the A‐helix (Juszczak et al, 2003). The conclusion followed that a change in the A‐helix packing is responsible for the decreased polymerization in E6V/E7A (Lesecq et al , 1996, 1997).…”

“…This alteration could also arise from a movement of the A‐helix relative to the E‐helix, different from that earlier demonstrated for HbC and HbS (Hirsch et al , 1999). This hypothesis was recently supported by a separate UVRR study (Juszczak et al, 2003) showing a strengthened Trp β15 (A12)–Ser β72 (E16) hydrogen bond and a tighter packing of the A‐helix against E‐helix for E6V/K132A.…”

Measurement of hemoglobin oxygen saturation using Raman microspectroscopy and 532-nm excitation

“…In the few studies where oxygenation was investigated, the RBCs were not studied under flowing conditions (45, 49 -51). Nevertheless, those studies demonstrate the potential for Raman spectroscopy in the study of RBC disorders, such as thalassemia, sickle-cell disease (21), and malaria (30).…”

Phase Separation and Crystallization of Hemoglobin C in Transgenic Mouse and Human Erythrocytes

“…The possibility that the complex RBC membrane (as well as some RBC cytosolic components) plays a role in HbC nucleation and crystallization was indicated earlier by in vitro studies demonstrating that oxy HbC crystallization is accelerated by the presence of the cytosolic N-terminus domain of human Band 3 and the 11 amino acid N-terminus Band 3 peptide (55). Oxy Hb binding to Band 3 may be explained by the following: oxy/liganded b6 mutant Hbs exhibit an altered central cavity that may give rise to their unique Hb intra-and intermolecular interactions (55)(56)(57)(58)(59). Nevertheless, with the data presented in this study, we cannot make any inference regarding the purported site of binding.…”