title: Role of hydrophobic amino acids at beta85 and beta88 in stabilizing F helix conformation of hemoglobin S.
abstract: Three Hb S variants containing Glu substitutions at Phe-beta85 and/or Leu-beta88 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 betaF85E,betaL88E was measured by CD and compared with those of Hb S betaF85E, Hb S betaL88E, 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 degrees C. However, changes in ellipticity at 222 nm for Hb S betaF85E in the CO form at 60 degrees C were about 15-fold greater than that of Hb S, while those for Hb S betaL88E and Hb S betaF85E,betaL88E 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 betaF85E,betaL88E was similar to that of Hb S betaL88E rather than Hb S betaF85E. These results suggest that Glu insertion at both beta85 and beta88 makes heme insertion into the heme pocket more difficult; however, once inserted, stability of Hb S betaF85E, betaL88E is similar to Hb S betaL88E rather than Hb S betaF85E. Furthermore, these results suggest that both Phe-beta85 and Leu-beta88 are critical for F helix stabilization and that Glu insertion at beta88 leads to more destabilization than insertion at beta85.
authors.affiliation: Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: L R
authors.initials: LR
authors.lastname: Reddy
authors.forename: K S
authors.initials: KS
authors.lastname: Reddy
authors.forename: S
authors.initials: S
authors.lastname: Surrey
authors.forename: K
authors.initials: K
authors.lastname: Adachi
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D018076:DNA, Complementary; D006451:Hemoglobin, Sickle / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D000596:Amino Acids; D018076:DNA, Complementary; D006451:Hemoglobin, Sickle; D011994:Recombinant Proteins