Role of Hydrophobic Amino Acids at β85 and β88 in Stabilizing F Helix Conformation of Hemoglobin S

Abstract

Three Hb S variants containing Glu substitutions at Phe-␤85 and/or Leu-␤88 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 ␤F85E,␤L88E was measured by CD and compared with those of Hb S ␤F85E, Hb S ␤L88E, 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 ␤F85E in the CO form at 60°C were about 15-fold greater than that of Hb S, while those for Hb S ␤L88E and Hb S ␤F85E,␤L88E 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 ␤F85E,␤L88E was similar to that of Hb S ␤L88E rather than Hb S ␤F85E. These results suggest that Glu insertion at both ␤85 and ␤88 makes heme insertion into the heme pocket more difficult; however, once inserted, stability of Hb S ␤F85E,␤L88E is similar to Hb S ␤L88E rather than Hb S ␤F85E. Furthermore, these results suggest that both Phe-␤85 and Leu-␤88 are critical for F helix stabilization and that Glu insertion at ␤88 leads to more destabilization than insertion at ␤85.In addition to Val-␤6, the key to polymerization of deoxy-Hb S is formation of a Val-␤6 hydrophobic acceptor pocket containing the hydrophobic amino acids Phe-␤85 and Leu-␤88 in the F helices of the T structure or deoxy form of Hb S which interacts with Val-␤6 (1, 2). Therefore, characterization of the Val-␤6 acceptor site, which includes Phe-␤85 and Leu-␤88, is important to understand hydrophobic interactions of deoxy-Hb S polymerization.We previously engineered and expressed Hb S variants containing Glu at either ␤85 or ␤88, Hb S ␤F85E, and Hb S ␤L88E and characterized their polymerization properties in order to understand the role of hydrophobicity and stereospecificity of the acceptor pocket during deoxy-Hb S polymerization (3). The deoxy form of Hb S ␤L88E polymerized in vitro at a much higher hemoglobin concentration than deoxy-Hb S containing other amino acid substitutions at ␤85 and ␤88 such as Hb S ␤F85E and Hb S ␤L88F (3). Furthermore, kinetics of polymerization for Hb S ␤L88E were biphasic at lower hemoglobin concentrations. Deoxy-Hb S ␤F85E also polymerized like deoxy-Hb S ␤L88E; however, the concentration required for polymerization of Hb S ␤F85E was 3-fold less than that of Hb S ␤L88E.These differences in polymerization between having Glu at ␤85 versus ␤88 may be due to residue location in the acceptor pocket. X-ray analysis of crystallized deoxy-Hb S shows that ␤85 and ␤88 amino acid side chains line the bottom of the acceptor pocket (1, 2), with Phe-␤85 located internally at the floor of the pocket and Leu-␤88 located near the pocket entrance. This difference in location might be expected to lead to different results when making the same Glu change at these two sites. Polymerization properties of several other recombinan...

Kex2-dependent invertase secretion as a tool to study the targeting of transmembrane proteins which are involved in ER-->Golgi transport in yeast.

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Identification of defensin binding to C1 complement

“…HNP-1, PG-1 and TP-1 were iodinated by the Iodo-gen or Iodobeads method [13] to a specific activity of 10 &i@g. Trace amounts of radiolabeled HNP-1, PG-1 or TP-1 was incubated with NHS, NHP, and purified Cl subcomponents for 30 min at 37'C. Resulting complexes were followed by native or rate PAGE at pH 5.7 [14] or at pH 8.8 [15]. After electrophoresis toward the anode in a Minigel apparatus (Hoeffer Scientific Instruments, San Francisco, CA) at 4"C, gels were Coomassie stained, dried and exposed to X-OMAT AR films (Eastman Kodak Co., Rochester, NY) at -70°C for l-3 days.…”

Site‐directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121

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