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Perutzll Institut National de la SantP et de la Recherche MPdicale, Unit& 299, H6pital de BicPtre. 94275 Le Kremlin-Bicdtre, France, Centre National de la Recherche Scientifique, Unit& de Recherche AssociPe 1 171, FacultP de MPdecine Grange-Blanche, 69373 zyxwvutsrqpon Lyon Cedex 08, France, and MRC Laboratory for Molecular Biology, Hills Road, Cambridge CB2 2QH, England Received January 19, 1990; Revised Manuscript Received April 9, 1990 ABSTRACT: We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the p chains, namely, H b Knossos or /3 A27S and Hb Grange-Blanche or p A27V. The p A27S mutant has a 50% decrease in oxygen affinity relative to native human Hb A, while the p A27V mutant has an increased oxygen affinity. We have also engineered the artificial p A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of H b A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the /3 chains; for the p A27S mutant a new hydrogen bond between serine and the carbonyl 0 at ,d 23 zyxwvu (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native H b A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities. xo hemoglobin (Hb)' natural variants have been reported recently at the /3 27 (B9) site, namely Hb Knossos (A27S) (Fessas et al., 1982; Baklouti et al., 1986) and Hb Grange- Blanche (A27V) (Baklouti et al., 1987). These mutants are of particular interest as they occur at the tight and highly hydrophobic cluster between the B and G helices in the /3 chains. X-ray analyses (Baldwin & Chothia, 1979; Shanaan, 1983; Fermi et al., 1984) have shown that in native Hb A this portion of the /3 chains does not undergo significant structural changes on oxygen binding. Substitution of the polar Ser residue for Ala in Hb Knossos leads to a 50% decrease in oxygen affinity; substitution of the more hydrophobic Val for Ala in Hb Grange-Blanche leads to an increased oxygen af- finity. To understand more precisely the reasons for the ab- normal oxygen affinities of these natural mutants, we have 'This work was supported by funds from Institut National de la Santt et de la Recherche Medicale, from La Direction des Recherches. Etudes et Techniques, from I'Air Liquide Co., and from La Fondation pour la Recherche MEdicale. * To whom correspondence should be addressed. ' Abbreviations: Hb, hemoglobin; DPG, 2,3-diphosphoglycerate; PSO, *lNSERM U 299. oxygen partial pressure at half-saturation; zyxw nS9' Hill coefficient at half- 8CNRS URA 1171. saturation; Bistris, bis(2-hydroxyethyl)aminotris(hydroxymethyI)- '1 MRC Laboratory for Molecular Biology. methane; HEPES, 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid. 0006-2960/90/0429-7020$02.50/0 0 1990 American Chemical Society