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Hemoglobin Einstein: Semisynthetic deletion in the B-helix of the -chain

¹ Departments of Medicine and of
² Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA
³ Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA

Reprint requests to: Seetharama A. Acharya, Departments of Medicine and of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; e-mail: acharya{at}aecom.yu.edu (718) 824-3153.

(RECEIVED December 12, 2003; FINAL REVISION February 3, 2004; ACCEPTED February 3, 2004)

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
The influence of the deletion of the tetra peptide segment _23–26 of the B-helix of the -chain of hemoglobin-A on its assembly, structure, and functional properties has been investigated. The hemoglobin with the deletion, ss-Hemoglobin-Einstein, is readily assembled from semisynthetic _1–141 des_23–26 globin and human ^A-chain. The deletion of _23–26 modulates the O₂ affinity of hemoglobin in a buffer/allosteric effector specific fashion, but has little influence on the Bohr effect. The deletion has no influence on the thermodynamic stability of the ₁₁ and the ₁₂ interface. The semisynthetic hemoglobin exhibits normal intersubunit interactions at the ₁₁ and ₁₂ interfaces as reflected by ¹H-NMR spectroscopy. Molecular modeling studies of ss-Hemoglobin-Einstein suggest that the segment _28–35 is in a helical conformation, while the segment _19–22 is the nonhelical AB region. The shortened B-helix conserves the interactions of ₁₁ interface. The results demonstrate a high degree of plasticity in the hemoglobin structure that accommodates the deletion of _23–26 without perturbing its overall global conformation.

Keywords: shortened B-helix; stuctural plasticity; thermodynamic stability; subunit interfaces; oxygen affinity; ¹H-NMR spectroscopy; molecular modeling

Abbreviations: ss-Hb, semisynthetic hemoglobin • HbA, human adult hemoglobin • RP-HPLC, reverse-phase high-performance liquid chromatography • kD, kilodaltons • HMB, p-hydroxymercuri benzoiate • NMR, nuclear magnetic resonance • DSS, dimethyl-2-silapentane 5-sulfonate • IEF, iso-electric focusing • HbCOA, carbon monoxy HbA • DPG, 2,3 diphosphoglyceric acid, IHP, inositol hexaphosphate • L-35, 3,5-dichloro phenylureido-phenoxy isobutyric acid • HEPES, N-(2-hydroxyethyl) piperazine -N-(2-ethane sulfonic acid)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03567804.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
The B-helix of both the - and -chains of Hb, which play a critical role in maintaining the tertiary and quaternary structure (Perutz 1970; Dickerson and Gies 1983), consist of 16 residues involving the amino acid residues 20 to 35 in the -chain and 19 to 34 in the -chain. The amino terminal region of the B-helix interacts with the GH-corner of the respective chains to establish the tertiary fold of the chains. On the other hand, the carboxyl terminal region of the B-helix plays an integral part in the quaternary structure of Hb by being part of the ₁₁ interface (Shaanan 1983; Fermi et al. 1984). The side chains of residues 31, 34, and 35 of the -chain interact with the last residue of the GH-region and the amino-terminal region of the H-helix of the -chain (residues 122, 123, 124, 125, 127, 128, and 131). Similar complementary interactions between the B-helix of the -chain with the GH-corner and the H-helix residues of the -chain exist. Residues 30, 33, and 34 of the B-helix of the -chain have noncovalent interactions with the last residue of the GH region and the amino-terminal region of the H-helix of the -chain (residues 117, 118, 119, 122, 123, and 126). Therefore, it is likely that the B-helix of both chains play an important role in preserving the integrity of the ₁₁ interface of Hb. A discontinuity between Glu₃₀ and Arg₃₁ of the -chain is permissible within the tertiary interactions of the chain (Seetharam and Acharya 1986; Seetharam et al. 1986). However, this discontinuity is not permissible within the constraints of the quaternary structure of Hb (Sahni et al. 1989).

The unusually high selectivity of the Glu-Arg peptide bond of the isolated -chain or -globin of Hb to V8 protease cleavage has been translated into a semisynthetic procedure (Sahni et al. 1989, 1991; Roy and Acharya 1994), permitting the splicing of _1–30, either synthesized chemically or generated from the slicing of the animal -globin, with either human or animal _31–141 to generate ss-chimeric -globin (Roy et al. 1993; Nacharaju et al. 1997; Srinivasulu et al. 1999; Rao et al. 2000). The chimeric -globins readily assemble with human ^A- or ^S-chains and generate functional tetramers containing chimeric -chains even when they carry a significant number of sequence differences in the amino-terminal region of the B-helix.

The interactions of the interfaces of Hb are coupled to the integrity of the central cavity of the protein, the region of the molecule that interacts with heterotropic effectors. Thus, the success in assembling tetramers carrying animal–human chimeric -chains (Roy et al. 1993; Nacharaju et al. 1997; Srinivasulu et al. 1999; Rao et al. 2000) with sequence differences at the amino-terminal region of the B-helix is a demonstration of a degree of flexibility in the tertiary and quaternary structure of Hb in terms of the amino acid residues that are compatible in the B-helix.

The structural plasticity of Hb is, apparently, responsible for the successful assembly of interspecies hybrid in vitro (Riggs and Herner 1962; Causegrove et al. 1984; Rao et al. 1996) as well as in the generation of interspecies hybrid Hbs in transgenic animals expressing HbA. The success in designing and generating Hb-Felix (Dumoulin et al. 1998) and Hb-Oscar (Tsai et al. 2001) exemplified the concept of structural plasticity in Hb. In Hb-Felix, the A helix of the -chain has been exchanged with that of the -chain while with Hb-Oscar, the reverse was achieved. In the tetramers of human ^A- or ^S-chains with swine–human chimeric -chain (_1–30 of swine spliced with _31–141 of human), the A-helix and part of the B-helix of human -chain of Hb has been exchanged with swine -chain (Rao et al. 2000). The results, therefore, suggest that chimeric B-helix (part swine and part human) is permissible in the assembly of the tetrameric structure of Hb. Accordingly, we seek to ask here whether the length of the B-helix of -chain itself could be shortened by deleting a contiguous set of four amino acid residues (one turn of the helix) without influencing the assembly of the overall Hb-fold when mixed with the -chain.

Recently, we have demonstrated that the deletion of the segment _23–26 from _17–30 has no influence on the equilibrium yield of the V8 protease catalyzed splicing of _17–30 with _31–40 (Srinivasulu and Acharya 2000, 2002). In the present study, we demonstrate that the deletion of _23–26 segment of _1–30 did not influence the V8 protease-catalyzed -globin splicing reaction. Also, the new ss--globin,_1–141des_23–26 (-Einstein globin), assembled with the ^A-chain to generate the tetrameric (₂₂) structure, Hb-Einstein ([_1–141des_23–26]₂₂^A), despite the fact that in the new ss--Einstein-chain, B-helix had been shortened by four amino acid residues (one turn). The functional and conformational consequences of the shortening of the B-helix of -chain are presented here.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Assembly and chemical characterization of ss-Hb-Einstein
Influence of internal deletion of _23–26 on the equilibrium yields of -globin semisynthetic reaction
The influence of deletion of _23–26 from _1–30 on its propensity to splice with _31–40 as well as with _31–141 is shown in Table 1. The product conformation-driven splicing of _17–30 and of _1–30 with _31–40 in the presence of V8 protease proceeds with an equilibrium yield of 42% (Srinivasulu and Acharya 2002). As shown in Table 1, replacing the carboxyl component either with _17–30des_23–26 or with _1–30des_23–26 has very little influence on the equilibrium yields. However, when _31–141 was used as the amino component instead of _31–40, the equilibrium yields of the splicing reaction (the generation of ss _1–141des_23–26) was reduced to nearly 30%. The deletion of the segment _23–26 of _1–30 lowers the splicing yield of Glu₃₀ with Arg₃₁ by about 25% compared to that with _1–30. This, presumably, reflects the lowered V8 protease resistance of the cosolvent induced secondary conformational features of ss--Einstein-globin relative to the parent -globin (Iyer and Acharya 1987).

View this table: [in this window] [in a new window]   Table 1. Influence of the deletion of 23–26 on the equilibrium yields in the V8 protease catalyzed splicing of the complementary segments of chain

View larger version (21K): [in this window] [in a new window]   Figure 1. Isolation of ss-Hb-Einstein. (A) Purification of semisynthetic -globin by CM-52 urea chromatography. Approximately 150 mg of mixture of semisynthetic 1–141des23–26 and 31–141 was loaded to the CM-52 column (2.5 x 15 cm), which was equilibrated with 5 mM phosphate buffer (pH 6.8) containing 8 M urea and 50 mM -mercaptoethonol. The column was eluted with a gradient between 25 and 50 mM phosphate buffer containing both 8 M urea and 50 mM -mercaptoethanol (250 mL each). Fractions were measured at 280 nm. Identification of the peaks was established by V8 protease digestion followed by the analysis on RP-HPLC using the C4 column. (B) Purification of ss-Hb-Einstein: CM-52 column (0.9 x 30 cm) equilibrated with 10 mM phosphate buffer (pH 6.5). Chromatogram was developed by a gradient between 10 mM phosphate buffer (pH 6.5) and 15 mM phosphate buffer (pH 8.3). A total of 500 mL of buffer was employed. Fractions were measured at 540 nm. Identification of the peaks was done by RP-HPLC analysis.

View larger version (27K): [in this window] [in a new window]   Figure 2. Analysis of the purity of ss-Hb-Einstein. An analytical fast-flow Q-Sepharose high-performance column was used to establish the purity of the ss-Hb-Einstein. The chromatograms were developed by a pH gradient using 50 mM Tris acetate buffer of pH 8.1 and 7.0, respectively, at a flow rate of 1 mL/min at 25°C. Inset A shows the RP-HPLC analysis of the ss-Hb-Einstein. Inset B compares the IEF pattern of ss-Hb-Einstein with that of HbA and HbS.

View this table: [in this window] [in a new window]   Table 2. Mass spectral analysis of ss--globin Einstein

Functional properties of ss-Hb-Einstein
The O₂ affinity of Hb in 50 mM Bis-Tris/50 mM Tris-acetate buffer, pH 7.4, at 37°C (Table 3; Fig. 3) is increased as a result of deletion of the tetra-peptide, _23–26; nonetheless, the O₂-binding to ss-Hb-Einstein is still cooperative, and is comparable to HbA (Fig. 3A). The Bohr effect of ss-Hb-Einstein is comparable to that of HbA (Fig. 3B).

View larger version (18K): [in this window] [in a new window]   Figure 3. Functional properties of ss-Hb-Einstein. (A) The cooperativity in the oxygen binding by ss-Hb-Einstein. The Hill plots for ss-Hb-Einstein and HbA are derived from the oxygenation curves (deoxy to oxy conformation) of the respective samples. These measurements were made using Hem-O-Scan at 37°C. Hemoglobin concentration was 0.5 mM. ss-Hb-Einstein (filled squares), HbA (open squares) in 50 mM Bis-Tris and 50 mM Tris acetate buffer (pH 7.4); ss-Hb- Einstein (filled triangles) and HbA (open triangles) 50 mM HEPES buffer (pH 7.4); and ss-Hb-Einstein (filled circles), HbA (open circles) in 100 mM phosphate buffer (pH 7.0). (B) The Bohr effect of ss-Hb-Einstein. Influence of the pH on the oxygen affinity of ss-Hb-Einstein and HbA are carried out using Hem-O-Scan at 37°C in 50 mM Bis-Tris and 50 mM Tris acetate buffer. These measurements were carried out over a pH range of 6.0 to 8.0 and the hemoglobin concentration was 0.5 mM. (Filled squares) ss-Hb-Einstein, and (open squares) HbA.

The O₂ affinity of ss-Hb-Einstein in the presence of allosteric effectors is given in Table 4. The deletion of _23–26 of HbA has no influence on the O₂ affinity in the presence of DPG, just as in the phosphate buffer. Similarly, in the presence of L-35, another allosteric effector (Lalezari et al. 1990), the O₂ affinity of ss-Hb-Einstein is comparable to that of HbA. On the other hand, ss-Hb-Einstein exhibits a higher O₂ affinity in the presence of IHP and sodium chloride (Cl^–). Thus, increase in the O₂ affinity of HbA resulting as a consequence of deletion of the segment _23–26 is not global; it is a buffer and allosteric effector-specific event.

Interdimeric interactions in ss-Hb-Einstein
The interactions at the interdimeric interface (₁₂ interface) of HbA are weaker in the oxy conformation relative to those in the deoxy conformation. The shortening of the B-helix of the -chain could influence the stability of the tetramer at the ₁₁ interface because the carboxyl end of B-helix is closely involved in the interactions of the interface, and it is likely that such a difference, if it exists, could be more pronounced in the oxy conformation.

The thermodynamic stability of the ₁₂ interface of the tetramer has been evaluated by size-exclusion chromatography on a Superose-12 column. HbA (loaded at a concentration of 0.5 mM) elutes from this column at 52 min (Fig. 4). A mutant Hb in which the ₁₂ interface has been weakened by the () Trp₃₇ Glu mutation, and that is known to exist only as a dimer, elutes from this column at 54 min, marking the elution position of dimer (MW 32 kD). The HMB -chain elutes around 59 min, marking the position of monomer (MW 16 kD). If the ₁₂ interface has been weakened in the ss-Hb-Einstein as a consequence of the shortening of the B-helix, and it exists primarily as a dimer, it will elute from the Superose-12 column at the position of the mutant HbA ([] W₃₇ E). ss-Hb-Einstein elutes from the Suprose-12 columns at 52.0 min. (Fig 4A), that is, at the same position as HbA. Therefore, the overall thermo-dynamic stability of the ₁₂ interface interactions in ss- Hb-Einstein appears to be comparable to that of HbA.

View larger version (17K): [in this window] [in a new window]   Figure 4. Interdimer interactions of ss-Hb-Einstein. The interaction of 12 interface of the ss-Hb-Einstein was established by size-exclusion chromatography on a Superose-12 column using AKTA. (A) The elution pattern of the ss-Hb-Einstein. (B) The elution profiles (Explorer 10 Purification System [Amersham Biosciences]) of HbA; r-HbA (HbAW37E) and pHMB-A-chain. All chromatograms were developed at 0.5 mL/min flow rate in phosphate buffered saline (pH 7.4).

Thermodynamic stability of the ₁₁ interface of ss-Hb-Einstein

The ss-Hb-Einstein was incubated with normal -chain of HbA to determine whether a weakening of the ₁₁ interface has occurred as a result of the deletion of the tetra peptide sequence _23–26 and, if so, whether the ss--Einstein chain of the tetramer can exchange for the free (un-hybridized) -chain in solution to generate HbA. Given the differences between the IEF patterns of HbA and ss-Hb-Einstein, the subunit exchange can be established by IEF analysis. The results of this analysis are shown in Figure 5. As can be seen, the -component of ss-Hb-Einstein does not exchange with the wild-type -chain. Hence, the interactions of ₁₁ interface in ss-Hb-Einstein are strong and do not permit the subunit exchange reaction. Thus, it is clear that ss-Hb-Einstein has normal interactions at the ₁₁ and ₁₂ interfaces and has an overall Hb-fold that is resistant to proteolysis with trypsin and V8 protease.

View larger version (30K): [in this window] [in a new window]   Figure 5. Subunit exchange with ss-Hb-Einstein. IEF pattern of ss-Hb-Einstein subjected to subunit exchange reaction for 2 h with normal human -chain. After incubation of the sample, the exchange of ss-Hb-Einstein with the human -chain was analyzed on IEF gels with a blend of pH 6.0–8.0 ampholytes. Lane 1, -chain; lane 2, ss-Hb-Einstein; lane 3, ss-Hb-Einstein + -chain; and lane 4, HbA.

View larger version (25K): [in this window] [in a new window]   Figure 6. 1H-NMR studies of ss-Hb-Einstein. (A) In CO form: 300 MHz 1H-NMR spectra of 5% of Hb A and ss-Hb-Einstein in the CO form in H2O in 0.1 M sodium phosphate at pH 7.0 and 29°C. (B) In the deoxy form: 300 MHz 1H-NMR spectra of 5% of Hb A and ss-Hb-Einstein in the deoxy form in H2O in 0.1 M sodium phosphate at pH 7.0 and 29°C.

The hyperfine-shifted and exchangeable proton resonances in the 300-MHz NMR spectra of HbA and ss-Hb-Einstein in the deoxy form are also shown in Figure 6B. The hyperfine-shifted N¹H resonances of proximal histidine of ss-Hb-Einstein in the deoxy form shown in the left panel are very similar to those of HbA. The hyperfine-shifted proton resonances arise from the protons on the heme groups and the nearby amino acid residues due to the hyperfine interactions between these protons and unpaired electrons of Fe (II) in the heme pocket (Ho 1992). In ss-Hb-Einstein, a small, noticeable change is seen in the microenvironment of -heme.

The exchangeable proton resonances of ss-Hb-Einstein in the deoxy form are compared with those of HbA in the right panel (Fig. 6B). The ¹H resonance at ~14 ppm has been identified as the intersubunit H-bond between Tyr₄₂ and Asp₉₉ in the ₁₂ interface in deoxy-HbA (Fung and Ito 1970), a characteristic feature of the deoxy (T)-quaternary structure of HbA (Perutz 1970). Another characteristic feature of the T structure is the appearance of the resonance at 11.4 ppm downfield from DSS, which is assigned to the H-bond between Trp₃₇ and Asp₉₄ in the ₁₂ interface (Fung and Ho 1975; Ho 1992; Ishimori et al. 1992). There are no noticeable differences in the resonances from 10–25 ppm between deoxy-HbA and deoxy-ss-Hb-Einstein. These results indicate that the internal deletion of _23–26 in the -chain of Hb does not alter the ₁₂ subunit interfaces in the deoxy form.

Molecular model of ss-Hb-Einstein
The consequence of the shortening of the B-helix by one turn on ss-Hb-Einstein has been investigated by molecular dynamic simulation, and is compared with that of HbA in Figure 7. The shortening of the B-helix of the -chain of HbA has a very limited influence on the global Hb-fold of the molecule (Fig. 7A). The deletion, however, has a pronounced influence on the B-helix itself (Fig. 7B). The segment _21–27des_23–26 of ss-Hb-Einstein has taken up a non-helical conformation, but the remaining region of the B-helix _28–35 appears to remain in helical conformation, and their position is not significantly different compared to that in the wild type. This is consistent with the NMR studies that the shortening of the B-helix does not influence the hydrogen-bonding pattern of the ₁₁ interface. The structure of Hb tetramer is able to tolerate the deletion of four residues of B-helix. This is achieved not by shortening the length of the B-helix by one turn but by altering the conformation of this region. In Hb-Einstein (only segment _28–35 remains helical), the amino terminal of the B-helix (₂₀–₂₇des_23–26) along with AB₁ residue (Ala₁₉) acquires a nonhelical conformation and acts as a linker between the shortened B-helix and the normal A-helix of the -chain. The molecular modeling studies have clearly reflected the plasticity in the Hb-fold, which is able to buffer changes in the helical regions by opening part of the original helical region into a nonhelical conformation to facilitate the given peptide segment to cover a longer distance to maintain the crucial interactions of the molecule.

View larger version (122K): [in this window] [in a new window]   Figure 7. Molecular model of ss-Hb-Einstein. The main-chain conformation of HbA and ss-Hb-Einstein has been depicted as a ribbon diagram in (A). The helical segments of the -chain are shown in green, and the -chain is shown in red except the B-helix of the -chain, which is shown in pink. Note that the part of the B-helix region of the -chain of ss-Hb-Einstein has assumed a nonhelical conformation as a result of the internal deletion of the residues 23 to 26. In HbA, the segment of the residues 23 to 26 of the B-helix of the -chain is shown in blue. This region is missing in ss-Hb-Einstein. Exploded views of the -chains of the two tetramers are shown in (B). In the -chain of HbA, the residues 23 to 26 that are deleted to generate ss-Hb-Einstein are marked in blue. As a consequence of this deletion, the region of residues 19 to 27 of the mutant -chain are now in a nonhelical conformation. This region as well as the rest of the B-helix is shown in pink.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

The present study extended this concept of the structural plasticity in Hb further, and established that -chain with internal deletions can be introduced semisynthetically at regions close to the intradimer interfaces. The studies have established that the -chain with an internal deletion of four residues in the B-helix (close to the region involved in the ₁₁ interface) generates a tetramer with normal structural stability comparable to that of HbA, binds O₂ reversibly, and responds to the presence of allosteric effectors. The internal deletion of four residues in the B-helix of the -chain represents a drastic structural change because it reduces the length of the B-helix. This region, together with the B-helix of the -chain, provides the basic scaffolding for the architecture of the Hb-tetramer.

The overall Hb-fold is conserved in ss-Hb-Einstein, as demonstrated by subunit exchange studies and the complete resistance of the tetramer to V8 protease and trypsin. The resistance to proteolysis reflects the strong interaction between -chain Einstein and ^A-chains of ss-Hb-Einstein. The resistance of Glu₃₀Arg₃₁ bond in the tetramer to V8 protease is particularly informative of the stability of the ₁₁ interface, because Arg₃₁ of the -chain is involved in the interaction of the ₁₁ interface and suggests that despite the shortened B-helix, the -Einstein chain and the ^A-chain generate normal subunit interfaces. NMR studies confirm the normal subunit interactions in the ss-Hb both in the oxy and deoxy states. The subunit exchange reaction demonstrates that the thermodynamics of the subunit interactions of HbA is also not jeopardized by the deletion of _23–26.

Consistent with these results, the consequences of molecular dynamic simulation studies demonstrate that the internal deletion on the conformation is localized. The molecule compensates the decreased length of the B-helix of the -chain by adopting a nonhelical conformation for the residues _19–27 of the molecule, and the rest of the B-helix residues _28–35 (B8 to B16) remain in the -helical conformation. As a result of the molecular rearrangement, ss-Hb-Einstein is able to maintain the normal subunit interactions of the tetramer. Thus, upon deletion of _23–26, the molecule conserves the overall Hb-fold by introducing a nonhelical AB-corner and shortened B-helix of eight residues instead of the original B-helix that was 16 residues long. Thus, this semisynthetic study has exposed the remarkable level of structural plasticity that exists in the Hb-fold.

Although ss-Hb-Einstein appears to conserve the overall Hb-fold, the functional studies reflect the influence of the deletion on the conformational dynamics of the protein in solution. The deletion of _23–26 increases the O₂ affinity of HbA. The near-normal O₂ affinity of ss-Hb-Einstein in the presence of allosteric effectors DPG and L-35 suggests that the influence of the shortened B-helix of ss-Hb-Einstein is communicated to both the and end of the central cavity. When the cognate allosteric effectors occupy the -and -ends, the influence of deletion is diminished. However, influence of deletion is not diminished in the presence of allosteric effectors IHP and Cl^–. The strong electrostatic modification of the -cleft that accompanies the binding of IHP to the -cleft suggests that ss-Hb-Einstein has become more sensitive to the electrostatic modifications relative to Hb, particularly in the oxy to deoxy conformational transitions.

The influence of the deletion of the segment _23–26 on the O₂ affinity of HbA is also diminished in 100 mM phosphate buffer. We have recently shown that the mid central cavity of HbA has two potential phosphate binding sites per -dimer (Acharya et al. 2003). An increase in the affinity of these sites to phosphate appears to occur as a consequence of Asn₁₀₈ ()-Lys (Hb-Presbyterian mutation), and the influence is also communicated to the -cleft. This molecular phenomenon appears to be responsible for the activation of the low O₂ affinity inducing potential of the Presbyterian mutation in the presence of Cl^– and phosphate (Acharya et al. 2003). It is conceivable that binding of the phosphate at these sites in the mid central cavity of Hb diminishes the influence of the shortening of the B-helix of the -chain on the -cleft.

The semisynthetic deletion was engineered in the middle of the B-helix of the -chain of HbA to shorten the length of the B-helix. However, the energetics of the Hb-fold did not permit the shortened B-helix to remain completely helical; it was reconfigured so that the carboxy terminal region of the shortened B-helix, namely the segment, _28–35, remains helical to facilitate the noncovalent interactions of the parent molecule, and the segment _19–27 remains nonhelical to connect the A-helix with the shortened B-helix. Thus, the crucial tertiary and/or quaternary interactions of the A and B-helix of the -chain are conserved. This reflects the delicate balance between the conformations of different regions of the Hb molecule, and the ability of modified regions to access a nonnative structure/conformation to favor a more generalized global Hb fold.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
Preparation of Hb samples
HbA was purified by DE-52 cellulose chromatography of the human red blood cell lysate followed by rechromatography on CM-52 cellulose column (Sahni et al. 1989). The - and -chains were separated by HMB reaction (Bucci 1981). Human -globin was generated by urea-CM-52 cellulose chromatography of the acid acetone precipitated globin (Roy and Acharya 1994).

Chemical synthesis of _1–30des_23–26
The peptide _1–30des_23–26 was chemically synthesized at the Macromolecular Chemistry Laboratory of Albert Einstein College of Medicine. The presence of the correct sequence was established by mass spectral analysis and tryptic mapping of the peptide.

V8 protease catalyzed splicing of _1–30des_23–26 with _31–40/_31–141
The protocol for splicing of _1–30des_23–26 with _31–40 was the same as that used for splicing of _17–30des_23–26 with _31–40 (Srinivasulu and Acharya 2002). For the semisynthesis of _1–141des_23–26, two equivalents of _1–30des_23–26 were incubated with one equivalent of _31–141 in 50 mM acetate buffer (pH 6.0) containing 30% propanol at 4°C. The splicing reaction was initiated by adding the V8 protease (1:200 w/w), and the progress was monitored by RP-HPLC for 48 h. The chromatogram was developed on an analytical Vydac C4 column using a gradient of 5% to 70% acetonitrile in 0.1% TFA. The splicing yield was calculated by redigesting the material eluting at the position of _31–141 with V8 protease and carrying out an RP-HPLC of the redigested material, because the spliced material does not resolve from _31–141 under the present chromatographic conditions.

The splicing reaction mixture containing the ss-_1–141des_23–26 (ss--globin Einstein) was lyophilized, and the unreacted _1–30des_23–26 was separated from ss -globin Einstein and the unreacted _31–141 by gel filtration on a Sephadex G-50 column (1.5 x 100 cm) equilibrated with 0.1% TFA. The unreacted _1–30des_23–26 is well resolved from the ss-_1–141des_23–26 on this column. The semisynthetic material coelutes with _31–141. The unreacted peptide and the semisynthetic product were pooled separately and lyophilized. The recovered unreacted peptide, _1–30des_23–26, was subjected to a second round of splicing reaction with a fresh batch of _31–141.

Purification of -globin-Einstein
The ss-_1–141des_23–26 is separated from unreacted _31–141 by CM-52 cellulose chromatography in the presence of 8 M urea (Roy and Acharya 1994). The chromatographic separation of _1–141des_23–26 is shown in Figure 1A. The peak fraction from each of the four UV absorbing peaks was subjected to V8 protease digestion (pH 4.0), and the digest was analyzed by RP-HPLC to identify _1–141des_23–26. The major peak that elutes between 420 and 450 mL was identified as the underivatized _31–141, and the peak eluting between 320 and 390 mL has been identified as _1–141des_23–26 (ss--globin Einstein). The fractions containing ss--globin Einstein are pooled and dialyzed against water in 0.1% acetic acid. The -globin variant, ss--globin Einstein, was lyophilized. The yield of ss--Einstein after the purification was about 20%–22% of the material loaded on the column.

Reconstitution of ss--Einstein with ^A-chains
The reconstitution of ss--globin Einstein with ^A-chain to generate the tetramers was achieved by alloplex intermediate pathway as described earlier (Roy and Acharya 1994). The reconstituted material was purified on CM-52 cellulose column (0.9 x 30 cm) with a pH gradient generated by 250 mL each of 10 mM phosphate buffer (pH 6.5) and 15 mM phosphate buffer (pH 8.3). The elution pattern of the protein was followed by measuring the absorbance of the effluent at 540 nm.

Functional and conformational studies of ss-Hb-Einstein
O₂ affinity of the Hb samples, susceptibility of the tetramer (ss-Hb-Einstein) to tryptic and V8 protease digestion, dimer–tetramer equilibrium, and subunit exchange reactions were carried out as described previously (Rao et al. 1996).

¹H-NMR spectroscopy
¹H-NMR spectra of HbA and ss-Hb-Einstein were obtained using a Bruker AVANCE DRX-300 spectrometer. All Hb samples were in 0.1 M sodium phosphate buffer (pH 7.0) in 100% water, and the Hb concentration was about 5% (~3 mM in heme). The water signal was suppressed by using a jump-and-return pulse sequence (Plateau and Gueron 1982). Proton chemical shifts were referenced to the methyl proton resonance of 2,2-dimethyl-2-silapen-tane-5-sulfonate (DSS) indirectly by the water signal that occurs at 4.76 ppm downfield from that of DSS at 29°C, as the internal reference.

Molecular modeling studies of ss-Hb-Einstein
The crystal structure of 4HHB [PDB] (Fermi et al. 1984) was chosen to model the truncation. The four residues 23, 24, 25, and 26 of ₁ and ₃ subunits of Hb were deleted one by one, and the perturbation of the structure was annealed by energy minimization followed by molecular dynamics at each stage using the program GROMACS (Berendsen et al. 1995; Lindahl et al. 2001). The truncated structure was placed in a box of water and then energy minimized with 1000 steps of steepest gradient method using the GROMACS force field using boundary conditions. Next the molecules were slowly heated to 300°C in steps of 100°C for 3 psec. The system was equilibrated at 300°C for 10 psec to derive the final structure. The figures were produced using the program Ras-MOl (Sayle and Milner-White 1995).

	Acknowledgments

 
This work was supported by NIH grants HL-24525, HL-38665, HL-55435, HL-58512, HL-58427, and HL-71064.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

	References

TOP Abstract Introduction Results Discussion Materials and methods References

 
Acharya, A.S., Malavalli, A., Peterson, E., Sun, D.P., Ho, C., Prabhakaran, M., Arnone, A., Manjula, B.N., and Friedman, J.M. 2003. Probing the conformation of hemoglobin Presbyterian in the R-state. J. Protein Chem. 22: 221–230.[CrossRef][Medline]

Berendsen, H.J.C., van der Spoel, D., and van Drunen, R. 1995. GROMACS: A message-passing parallel molecular dynamics implementation. Comp. Phys. Commun. 91: 43–56.[CrossRef]

Bucci, E. 1981. Preparation of isolated chains of human hemoglobin. Methods Enzymol. 76: 97–105.[Medline]

Causgrove, T., Goss, D.J., and Parkhurst, L.J. 1984. Oxygen equilibrium studies on carp-human hybrid hemoglobins. Biochemistry 23: 2168–2173.[CrossRef][Medline]

Chang, C.K., Simplaceanu, V., and Ho, C. 2002. Effects of amino acid substitutions at 131 on the structure and properties of hemoglobin: evidence for communication between 1 1- and 1 2-subunit interfaces. Biochemistry. 41: 5644–5645.[CrossRef][Medline]

Dalvit, C. and Ho, C. 1985. Proton nuclear Overhauser effect investigation of the hemepockets in ligated hemoglobin: Conformational differences between oxy and carbonmonoxy forms. Biochemistry 24: 3398–3407.[CrossRef][Medline]

Dickerson, R.E. and Geis, I. 1983. Hemoglobin: Structure, function, evolution and pathology. Benjamin/Cummings, Menlo Park.

Dumoulin, A., Padovan, J.C., Manning, L.R., Papowicz, A., Winslow, R.M., Chait, B.T., and Manning, J.M. 1998. The N-terminal sequence affects distant helix interactions in hemoglobin. Implications for mutant proteins from studies on recombinant hemoglobin Felix. J. Biol. Chem. 273: 35032–35038.[Abstract/Free Full Text]

Fermi, G., Perutz, M.F., Shaanan, B., and Fourme, R. 1984. The crystal structure of human deoxy haemoglobin at 1.74 A^O resolution. J. Mol. Biol. 175: 159–174.[CrossRef][Medline]

Fung, L.W.-M. and Ho, C. 1975. A proton nuclear magnetic resonance study of the quaternary structure of human hemoglobins in water. Biochemistry 14: 2526–2535.[CrossRef][Medline]

Ho, C. 1992. Proton nuclear magnetic resonance studies of hemoglobin: Cooperative interactions and partially ligated intermediates. Adv. Protein Chem. 43: 153–312.[Medline]

Huisman, T.H.J., Wilson, J.B., Gravely, M., and Hubbard, M. 1974. Hemoglobin Grady: The first example of a variant with elongated chains due to an insertion of residues. Proc. Natl. Acad. Sci. 71: 3270–3273.[Abstract/Free Full Text]

International Hemoglobin Information Center. 1995. Variant List (compiled by M.F.H. Carver and A. Kutlar). Hemoglobin 19: 39–124.

Ishimori, K., Imai, K., Miyazaki, G., Kitagawa, T., Wada, Y., Morimoto, H., and Morishima, I. 1992. Site-directed mutagenesis in hemoglobin: Functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37 and 145 mutations. Biochemistry 31: 3256–3264.[CrossRef][Medline]

Iyer, K.S. and Acharya, A.S. 1987. Conformational studies of -globin in 1-propanol: Propensity of the alcohol to limit the sites of proteolytic cleavage. Proc. Natl. Acad. Sci. 84: 7014–7018.[Abstract/Free Full Text]

Kawata, R., Ohba, Y., Yamamoto, K., Miyaji, T., Makita, R., Ohga, K., Watanabe, S., and Miwa, S. 1988. Hyperunstable hemoglobin Koriyama; Anti-Hb Gun Hill insertion of five residues in the -chain. Hemoglobin 12: 311–321.[Medline]

Lalezari, I., Lalezari, P., Poyart, C., Marden, M., Kister, J., Bohn, B., Fermi, G., and Perutz, M.F. 1990. New effectors of human hemoglobin: Structure and function. Biochemistry 29: 1515–1523.[CrossRef][Medline]

Lindahl, E., Hess, B., and van der Spoel, D. 2001. GROMACS 3.0: A package for molecular simulation and trajectory analysis. J. Mol. Mod. 7: 306–317.

Lindstrom, T.R., Noren, I.B.E., Charache, S., Lehmann, H., and Ho, C. 1972. Nuclear magnetic resonance studies of hemoglobins. VII. Tertiary structure around ligand binding site in carbomonaxyhemoglobin. Biochemistry 11: 1677–1681.[CrossRef][Medline]

Nacharaju, P., Roy, R.P., White, S.P., Nagel, R.L., and Acharya, A.S. 1997. Inhibition of sickle -chain (^S)-dependent polymerization by nonhuman -chains. A superinhibitory mouse-horse chimeric -chain. J. Biol. Chem. 272: 27869–27876.[Abstract/Free Full Text]

Perutz, M.F. 1970. Sterio chemistry of cooperative effects in hemoglobin. Nature 228: 726–739.[CrossRef][Medline]

Plateau, P. and Gueron, M. 1982. Exchangeble proton NMR without base-line distortion using new strong-pulse sequence. J. Am. Chem. Soc. 104: 7310–7311.[CrossRef]

Rao, M.J., Manjula, B.N., Kumar, R., and Acharya, A.S. 1996. Chimeric hemoglobins-hybrids of human and swine hemoglobin: Assembly and stability of inter species hybrids. Protein Sci. 5: 956–965.[Abstract]

Rao, M.J., Malavalli, A., Manjula, B.N., Kumar, R., Prabhakaran, M., Sun, D.P., Ho, N.T., Ho, C., Nagel, R.L., and Acharya, A.S. 2000. Interspecies hybrid HbS: Complete neutralization of val 6()-dependent polymerization of human -chain by pig -chains. J. Mol. Biol. 300: 1389–1406.[CrossRef][Medline]

Riggs, A. and Herner, A.E. 1962. The hybridization of donkey and mouse hemoglobins. Proc. Natl. Acad. Sci. 48: 1664–1670.[Free Full Text]

Roy, R.P. and Acharya, A.S. 1994. Semisynthesis of hemoglobin. Methods Enzymol. 231: 194–215.[Medline]

Roy, R.P., Nagel, R.L., and Acharya, A.S. 1993. Molecular basis of the inhibition of ^S-chain-dependent polymerization by mouse- chain. Semisynthesis of chimeras of human and mouse -chains. J. Biol. Chem. 268: 16406–16412.[Abstract/Free Full Text]

Russu, I.M., Ho, N.T., and Ho, C. 1987. A proton nuclear overhauser effect investigation of the subunit interfaces in human normal and adult hemoglobin. Biochim. Biophys. Acta 914: 40–48.[CrossRef][Medline]

Sahni, G., Cho, Y.J., Iyer, K.S., Khan, S.A., Seetharam, R., and Acharya, A.S. 1989. Semisynthetic hemoglobin A: Reconstitution of functional tetramers from semisynthetic globin. Biochemistry 28: 5456–5461.[CrossRef][Medline]

Sahni, G., Mallia, A.K., and Acharya, A.S. 1991. Proteosynthetic activity of immobilized staphylococcus aureus V8 protease: Application in the semisynthesis of molecular variants of -globin. Anal. Biochem. 193: 178–185.[CrossRef][Medline]

Sayle, R.A. and Milner-White, E.J. 1995. RASMOL: Biomolecular graphics for all. Trends Biochem. Sci. 20: 374–376.[CrossRef][Medline]

Seetharam, R. and Acharya, A.S. 1986. Synthetic potential of Staphylococcus aureus V8– protease: An approach toward semisynthesis of covalent analogs of -chain of hemoglobin S. J. Cell Biochem. 30: 87–99.[CrossRef][Medline]

Seetharam, R., Dean, A., Iyer, K.S., and Acharya, A.S. 1986. Permissible discontinuity region of the chain of hemoglobin: Noncovalent interactions of heme and the complementary fragments _1–30 and _31–141. Biochemistry 25: 5949–5955.[CrossRef][Medline]

Shaanan, B. 1983. Structure of human oxyhaemoglobin at 2.1 A^O resolution. J. Mol. Biol. 171: 31–59.[Medline]

Simplaceanu, V., Lukin, J.A., Fang, T.-Y., Zou, M., Ho, N.T., and Ho, C. 2000. Chain-selective isotopic labeling for NMR studies of large multimeric proteins: Application to hemoglobin. Biophys. J. 79: 1146–1154.[Abstract/Free Full Text]

Srinivasulu, S. and Acharya, S.A. 2000. Proteolytic splicing of Hb -chain with internal deletions. Proc. 16^th Am. Pep. Symp.