ABSTRACT: Three new Hb S variants containing β87 Leu, Trp, or Asp instead of Thr were expressed in yeast in order to further define the role of the β87 position in stability and polymerization of deoxy Hb S. Previous studies showed that hydrophobicity at β85 Phe and β88 Leu is critical for stabilization of hemoglobin. Results with the three Hb S β87 variants, however, showed minimal differences in stability, suggesting that β87 amino acid hydrophobicity is not critical for stabilization of hemoglobin. Polymerization properties of the variants in the deoxy form, however, were affected by the β87 amino acid. Polymerization of Hb S β87 Thr → Leu and Hb S β87 Thr → Trp was preceded by a delay time like Hb S, while Hb S β87 Thr → Asp did not show a delay time. In addition, changes in time required for half polymer formation (T1/2) as a function of hemoglobin concentration for Hb S β87 Thr → Asp were similar to that for β87 Thr → Gln. Hb S β87 Thr → Leu polymerized at a lower hemoglobin concentration than Hb S while β87 Thr → Trp and Hb S β87 Thr → Asp required much higher hemoglobin concentrations for polymer formation. Critical concentration required for deoxy Hb S β87 Thr → Asp polymerization was 6- and 2.3-fold greater than that for Hb S β85 Phe → Glu and Hb S β88 Leu → Glu, respectively. These results suggest that even though β87 Thr is not a direct interaction site for β6 Val in deoxy Hb S polymers, it does play a critical role in formation of the hydrophobic acceptor pocket which then promotes protein−protein interactions facilitating formation of stable nuclei and polymers of deoxy Hb S.
ABSTRACT: To understand determinants for hemoglobin (Hb) stability and Hb A2 inhibition of Hb S polymerization, three Valδ6 Hb A2 variants (Hb A2 δE6V, Hb A2 δE6V,δQ87T, and Hb A2 δE6V,δA22E,δQ87T) were expressed in yeast, and stability to mechanical agitation and polymerization properties were assessed.
Oxy forms of Hb A2 δE6V and Hb A2 δE6V,δQ87T were 2- and 1.6-fold, respectively, less stable than oxy-Hb S, while the stability of Hb A2 δE6V,δA22E,δQ87T was similar to that of Hb S, suggesting that Alaδ22 and Glnδ87 contribute to the surface hydrophobicity of Hb A2. Deoxy Hb A2 δE6V polymerized without a delay time, like deoxy Hb F γE6V, while deoxy Hb A2 δE6V,δQ87T and deoxy Hb A2 δE6V,δA22E,δQ87T polymerized after a delay time, like deoxy Hb S, suggesting that β87 Thr is required for the formation of
nuclei. Deoxy Hb F γE6V,γQ87T showed no delay time and required a 3.5-fold higher concentration than deoxy Hb S for polymerization,
suggesting that Thr effects on Valδ6 Hb A2 and Valγ6 Hb F variants are different. Mixtures of deoxy Hb S/Hb A2 δE6V,δQ87T polymerized, like deoxy Hb S, while polymerization of Hb S/Hb A2 δE6V mixtures was inhibited, like Hb S/Hb F γE6V mixtures. These results suggest α2βSδ6 Val, 87 Thr hybrids and Hb A2 δE6V,δQ87T participate in Hb S nucleation, while only 50% of α2βSδ6 Val hybrids and none of the Hb A2 δE6V participate. These findings are in contrast to those of mixtures of Hb S with Hb F γE6V or Hb F γE6V,Q87T, which both
inhibit Hb S polymerization. Our results also suggest participation in nucleation of some α2βSδ hybrids in A2S mixtures but not α2βSγ hybrids in FS mixtures.
Journal of Biological Chemistry 10/1996; 271(40):24557-24563. · 4.77 Impact Factor
ABSTRACT: Three Hb S variants containing Glu substitutions at Phe-β85 and/or Leu-β88 were expressed in yeast in an effort to evaluate
the role of hydrophobic amino acids at these sites in stabilizing F helix conformation of Hb S. Helix stability of tetrameric
Hb S βF85E,βL88E was measured by CD and compared with those of Hb S βF85E, Hb S βL88E, Hb A, and Hb S. The CD spectra of these
Hb S variants were similar to those of Hb S and Hb A at 10°C. However, changes in ellipticity at 222 nm for Hb S βF85E in
the CO form at 60°C were about 15-fold greater than that of Hb S, while those for Hb S βL88E and Hb S βF85E,βL88E were similar
and about 30-fold greater than Hb S. Thermal stability measured by continuous scanning of spectral changes revealed the three
Hb S variants were much more unstable than Hb S, and stability of Hb S βF85E,βL88E was similar to that of Hb S βL88E rather
than Hb S βF85E. These results suggest that Glu insertion at both β85 and β88 makes heme insertion into the heme pocket more
difficult; however, once inserted, stability of Hb S βF85E,βL88E is similar to Hb S βL88E rather than Hb S βF85E. Furthermore,
these results suggest that both Phe-β85 and Leu-β88 are critical for F helix stabilization and that Glu insertion at β88 leads
to more destabilization than insertion at β85.
Journal of Biological Chemistry 10/1996; 271(40):24564-24568. · 4.77 Impact Factor
ABSTRACT: Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at β85 were made and expressed in yeast in an effort to evaluate the role of Phe-β85 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 β385 substitutions do not significantly affect heme-globin interactions and tetramer helix content. Hb S containing Trp-β85 had decreased oxygen affinity, whereas those with Leu-, Ala-, and Thr-β85 had increased oxygen affinity. All four supersaturated β85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe-β88, Ala-β88, Glu-β88, or Glu-β85, 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 β85 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 β85 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 SFβ85L, Hb SFβ85A, Hb SFβ85T, and Hb sFβ85W are 0.65-, 2.2-, 2.5- and 3-fold higher, respectively, than Hb S. These results suggest that the relative order for polymerization of β85 variants (Leu > Phe > Ala > Thr > Trp-β85) depends on amino acid hydrophobicity rather than stereospecificity of the side chain. These findings are in contrast to previous results for β88 variants. Trp-β85 in Hb S may affect Val-β6 acceptor pocket size, but may still accommodate insertion of Val-β6. These results also strengthen our previous conclusion that β88 amino acid stereospecificity is more critical than that of β85 for insertion of β6 Val.
Protein Science 06/1995; 4(7):1272 - 1278. · 2.80 Impact Factor