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Mutational analysis of phenylalanine beta 85 in the valine beta 6 acceptor pocket during hemoglobin S polymerization
Abstract:
Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at beta 85 were made and expressed in yeast in an effort to evaluate the role of Phe-beta 85 in the acceptor pocket during polymerization of deoxy Hb S. The four Hb S variants have the same electrophoretic mobility as Hb S, and these beta 85 substitutions do not significantly affect heme-globin interactions and tetramer helix content. Hb S containing Trp-beta 85 had decreased oxygen affinity, whereas those with Leu-, Ala-, and Thr-beta 85 had increased oxygen affinity. All four supersaturated beta 85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe-beta 88, Ala-beta 88, Glu-beta 88, or Glu-beta 85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477-17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563-31566). Leu substitution at beta 85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization. The length of the delay time and total polymer formed for these beta 85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed. Critical concentrations required for polymerization of deoxy Hb SF veta 85L, Hb SF beta 85A, Hb SF beta 85T, and Hb SF beta 85W are 0.65-, 2.2-, 2.5- and 3-fold higher, respectively, than Hb S.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Substitutions at beta 85 of hemoglobin S (Hb S) influence polymerization. Leu substitution accelerated polymerization, while Ala, Thr, and Trp inhibited it, affecting deoxy Hb S polymerization dynamics.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Sickle cell disease (SCD) is caused by a mutation in hemoglobin S (Hb S).
- The polymerization of deoxy Hb S is a key event in SCD pathogenesis.
- Understanding the structural factors that modulate Hb S polymerization is crucial for developing therapies.
Purpose of the Study:
- To investigate the role of phenylalanine at position beta 85 (Phe-beta 85) in the acceptor pocket during deoxy Hb S polymerization.
- To evaluate the effects of specific amino acid substitutions at beta 85 on Hb S polymerization kinetics and oxygen affinity.
Main Methods:
- Hemoglobin S variants with Leu, Ala, Thr, or Trp substitutions at beta 85 were engineered and expressed in yeast.
- Electrophoretic mobility, heme-globin interactions, and tetramer helix content were analyzed.
- Oxygen affinity and polymerization kinetics (delay time, polymer formation) of the engineered Hb S variants were measured.
Main Results:
- The beta 85 substitutions did not significantly alter heme-globin interactions or tetramer helix content.
- Trp-beta 85 reduced oxygen affinity; Leu-, Ala-, and Thr-beta 85 increased it.
- All four variants exhibited polymerization delay, unlike some previously studied beta 88 variants.
- Leu substitution accelerated polymerization, while Ala, Thr, and Trp inhibited it, with varying critical concentrations required for polymerization.
Conclusions:
- Amino acid substitutions at beta 85 significantly modulate deoxy Hb S polymerization.
- The nature of the substitution at beta 85 dictates whether polymerization is accelerated or inhibited.
- These findings provide insights into the structural determinants of Hb S polymerization and potential therapeutic targets.