The N-terminal Sequence Affects Distant Helix Interactions in Hemoglobin

Abstract

The N-terminal 18-amino acid sequence of the ␤-chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the ␥-chain of fetal hemoglobin with the remaining sequence of the ␤-chain retained (helices B through H). The ␥-␤-chain had the correct mass, and its entire sequence was established by mass spectrometric analysis of its tryptic peptides; the ␣-chain also had the correct mass. This recombinant hemoglobin (named Hb Felix) retains cooperativity and has an oxygen affinity like that of HbA both in the presence and absence of the allosteric regulators, 2,3-diphosphoglycerate or chloride but differs from HbF in its 2,3-diphosphoglycerate response. However, Hb Felix has some features that resemble fetal hemoglobin, i.e. its significantly decreased tetramerdimer dissociation and its circular dichroism spectrum, which measure the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively. Even though Hb Felix contains the HbA amino acids at its tetramer-dimer interface, which is located at a distance from the substitution sites, its interface properties resemble those of HbF. The amino acid sequence of a protein contains the information necessary for its proper folding into the native state (1). Predicting patterns of protein folding into secondary and tertiary structures based on the primary structure is currently the focus of intense study in many laboratories. Assembly of such folded polypeptides into specific quaternary structures depends on how the individual subunits interact with one another. In human tetrameric hemoglobins there are two quaternary conformations, oxy (R) and deoxy (T), which are responsible for its functional binding and release of O 2 , respectively (2, 3). We describe here how some of these quaternary subunit interactions involving ␣ subunits in adult (␣ 2 ␤ 2 ) and fetal (␣ 2 ␥ 2 ) hemoglobins are influenced by the N-terminal sequences of their ␤ and ␥ subunits.Adult and fetal hemoglobins have several significant differences in their functional behavior at the quaternary level. These differences include their responses to DPG 1 binding, which affect their ability to carry O 2 (4), the strengths of their tetramer-dimer interactions as recently reported (5), and the circular dichroism and absorption spectra of the aromatic amino acids in their polypeptide chains (6). These well defined and accurately measured properties in adult and fetal hemoglobins are due to unique subunit interactions in the ␣ 2 ␤ 2 and the ␣ 2 ␥ 2 tetramer, respectively, that originate from the 39 amino acid substitutions between the ␤-and ␥-chains. Therefore, comparative studies of these hemoglobins are potentially informative in establishing a relationship between amino acid sequence and quaternary conformational properties.The tetramer-dimer interface of fetal hemoglobin undergoes dissociation about 70 times less readily than the corresponding interface in adult hemoglobin (5). In an effort to explain thi...

Linkage of Interactions in Sickle Hemoglobin Fiber Assembly

“…A similar trend is also apparent in the C␣-C␣ distance distribution calculated for the Ala 13 -Leu 113 pair. In the case of the Lys 16 -Glu 116 and Lys 16 -Leu 113 pairs, the effect is less pronounced for the C␣-C␣ distances, but is clearly visible when one considers the side chain atoms NZ of Lys 16 and CD of Glu 116 (data not shown). We used dynamical cross-correlation maps to identify corre- lated movements of residues from MD simulations (36,37).…”

“…10) was found to be 17 mg/ml. Using identical assay conditions, we had previously reported a C sat of about 39 mg/ml for the HbS-carrying point mutation of ␣Lys 16 3 Gln as against 30 mg/ml for native HbS (24). Thus, the presence of His 78 in the double mutant not only annulled the inhibitory effect due to mutation at ␣16 site but also conferred significant polymerization-enhancing propensity to the double-mutant HbS.…”

“…Hemoglobin tetramers in each strand are connected through a network of axial contacts (4). These contacts involve AB and GH regions of both the ␣-and ␤-chains that include ␣Lys 16 The presence of a large number of contacts at the interface between the two HbS molecules in each strand of the fiber raises the intriguing possibility that even subtle changes in the conformational dynamics generated because of the perturbation of non-contact residues around AB or GH regions of the chains might influence the polymerization process by interfering with inter-tetrameric interaction interface of the fiber.…”

“…The shift in C sat of the triple mutant by about 17 mg/ml ((45Ϫ28) mg/ml) clearly suggests that the polymerization-enhancing effects due to the presence of His 78 at the EF corner synergistically compensate for the inhibitory effect emanating from the Gly substitutions at the AB corner. 16 in which His 78 mutation was coupled to mutation of a known intra-double-strand contact site at ␣16. The C sat of the above double mutant (Fig.…”

“…Deoxygenated sickle hemoglobin (HbS) polymerizes into long helical fibers that are believed to be responsible for the pathophysiology of the sickle cell disease. The knowledge gleaned so far from structural analysis of HbS crystal (2)(3)(4), solution polymerization studies of natural variants or engineered mutant hemoglobins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24), and electron microscopic studies (25) have led to a 14-stranded model of the fiber (26,27). These strands appear as seven double strands of the type found in HbS crystals, albeit with a slight twist caused by fiber packing.…”