ABSTRACT
X-ray crystallographic studies indicate that the hydrophobic acceptor pocket made by E and F helices involving Leu-beta 88 and Phe-beta 85 is critical for the formation of stable hydrophobic interactions with Val-beta 6 on an adjacent deoxy-hemoglobin (Hb) S tetramer. Ala and Phe substitutions at the beta 88 position in Hb S were made using a yeast expression system in an effort to clarify the role of Leu-beta 88 in creating a suitable acceptor site for Val-beta 6 during polymerization of Hb S. Both Ala- and Phe-beta 88 substitutions in Hb S inhibited polymerization compared with Hb S. Critical concentrations for polymerization of alpha 2 beta 2 Val-6,Ala-88 and alpha 2 beta 2Val-6,Phe-88 were 6- and 10-fold higher, respectively, than that of Hb S (alpha 2 beta 2Val-6,Leu-88). Deoxy-Hb S containing Phe-beta 88 polymerized without a delay time like Trp-beta 6- and Phe-beta 6-substituted hemoglobins (Adachi, K., Konitzer, P., Kim, J., Welch, N., and Surrey, S. (1993) J. Biol. Chem. 268, 21650-21656). In contrast, oversaturated deoxy-Hb S containing Ala-beta 88 also polymerized without a delay time; however, with decreasing hemoglobin concentrations, the kinetics of polymerization were biphasic. At lower hemoglobin concentrations, closer to the critical concentration for polymerization, deoxy-Hb S containing Ala-beta 88 polymerized after a distinct delay time. These results suggest that bulky beta 88 hydrophobic replacements like Phe may sterically inhibit insertion of Val-beta 6 into the acceptor pocket. In contrast, smaller sized, less hydrophobic amino acids like Ala compared with Leu-beta 88 may allow insertion of Val-beta 6 into the acceptor pocket but may not promote stable protein-protein interactions with an adjacent Hb molecule. Stereospecificity and hydrophobicity of the Val-beta 6 hydrophobic acceptor pocket as well as the beta 6 amino acid are, therefore, critical for polymerization of deoxy-Hb S.