Conformational changes in hemoglobin S (βE6V) imposed by mutation of the βGlu⁷-βLys¹³² salt bridge and detected by UV resonance raman spectroscopy

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, βGlu⁷-βLys¹³², are dependent on which residue of the pair is modified. The βK132A mutants exhibit the spectroscopic signatures of the R → T state transition in both the "hinge" and "switch" regions of the α₁β₂ interface. Both singly and doubly mutated hemoglobin (Hb) βE7A exhibit the switch region signature for the R → 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 βE7A mutants. The observed large decrease in the W3 α14β15 band intensity for doubly mutated Hb βE7A is attributed to an enhanced separation in the A helix-E helix tertiary contact of the β subunits. The results for the Hb A βGlu⁷-βLys¹³² salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the α₁β₂ 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.