Site-directed mutagenesis in hemoglobin: functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37.beta. and 145.beta. mutations

Abstract

In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37 beta, in which Trp-37 beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94 alpha and Trp-37 beta at the alpha 1-beta 2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, 1H-NMR data indicated that the structure of deoxy Hb Phe-37 beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37 beta----Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37 beta suggested that a large part of the abnormal properties of Hb Phe-37 beta observed for dilute solutions appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94 alpha and Trp-37 beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145 beta has a Phe residue at the 145 beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145 beta and the carbonyl group of Val-98 beta in deoxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

“…The signals at 14.1 and 11.2 ppm have been assigned to the hydrogen bonds between ␣Tyr-42 and ␤Asp-99 and between ␤Trp-37 and ␣Asp-94, respectively. These residues are located in the ␣ 1 ␤ 2 interface, and the resonances are important T-state markers of the deoxy-Hb A (39,46,47). We did not detect any difference in chemical shift for these signals among the five proteins.…”

Substitution of the Heme Binding Module in Hemoglobin α- and β-Subunits

“…Asp(G1) (35) and ␣1 Asp(G1)-␤2 Trp(C3), respectively (38). The deoxy-␣ 2 [␤␣(HBM) 2 ] tetramer also afforded these additional peaks, although the peak positions slightly moved from the positions for Hb A (Fig.…”

“…In the far downfield region, the hyperfineshifted proton signals from the proximal histidine are observed for hemoglobin subunits and tetrameric Hb A (Fig. 4A) (38,39) In deoxygenated tetrameric Hb A, the proximal histidyl N ␦ H protons in the ␣-and ␤-subunits are detected at 64 and 76 ppm, respectively. The (40), the similar spectral pattern indicates that heme electronic state of Hb A did not significantly change upon the substitution of the heme binding module.…”

Assembly of Human Hemoglobin

“…(G1) at the ␣ 1 ␤ 2 interface (25,26); the resonance at 13.0 ppm to the hydrogen bond between Asp ␣126 (H9) and Tyr ␤35 (C1) at the ␣ 1 ␤ 1 interface; the resonance 12.2 ppm to the hydrogen bond between His ␣103 (G10) and Asn ␤108 (G10) at the ␣ 1 ␤ 1 interface (26); and the resonance at 11.1 ppm to the hydrogen bond between (C3) at the ␣ 1 ␤ 2 interface (27). 3 As shown in the figure, in deoxy-␣rHbA these four exchangeable proton resonances are very close if not identical to those in deoxy-HbA.…”