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• W2022251609 abstract "The AB and GH regions of the α-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala19 and Ala21 of the AB corner with more flexible Gly or rigid α-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of α-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (αAsn78 → His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of αGln78, His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (αGln78 → His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His78. Similar synergistic coupling, between αHis78 and an intra-double-strand contact point (α16) mutation located in the AB region, was also observed. Thus, two conclusions are made: (i) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and (ii) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with α78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly. The AB and GH regions of the α-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala19 and Ala21 of the AB corner with more flexible Gly or rigid α-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of α-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (αAsn78 → His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of αGln78, His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (αGln78 → His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His78. Similar synergistic coupling, between αHis78 and an intra-double-strand contact point (α16) mutation located in the AB region, was also observed. Thus, two conclusions are made: (i) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and (ii) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with α78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly. Sickle cell anemia arises because of a point mutation at the sixth position of the β-chain (βGlu6 → Val) in the hemoglobin (Hb) 1The abbreviations used are: Hb, hemoglobin; HbS, sickle Hb; Aib, α-aminoisobutyric acid; HL, Hanuman langur; RP-HPLC, reverse-phase high-performance liquid chromatography; ESMS, electro-spray mass spectrometry; FPLC, fast protein liquid chromatography; MD, molecular dynamics; RMSF, root-mean-squared fluctuations; BPG, bisphosphoglycerate. molecule (1Ingram V.M. Nature. 1956; 178: 792-794Crossref PubMed Scopus (478) Google Scholar). 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 (2Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8272-8279Abstract Full Text PDF PubMed Google Scholar, 3Padlan E.A. Love W.E. J. Biol. Chem. 1985; 260: 8280-8291Abstract Full Text PDF PubMed Google Scholar, 4Harrington D.J. Adachi K. Royer Jr., W.E. J. Mol. Biol. 1997; 272: 398-407Crossref PubMed Scopus (132) Google Scholar), solution polymerization studies of natural variants or engineered mutant hemoglobins (5Bookchin R.M. Nagel R.L. Ranney H.M. J. Biol. Chem. 1967; 242: 248-255Abstract Full Text PDF PubMed Google Scholar, 6Bookchin R.M. Nagel R.L. Semin. Hematol. 1974; 11: 577-595PubMed Google Scholar, 7Benesch R.E. Kwong S. Benesch R. Edalji R. Nature. 1977; 269: 772-775Crossref PubMed Scopus (44) Google Scholar, 8Bookchin R.M. Balaz T. Nagel R.L. Tellez I. Nature. 1977; 269: 526-527Crossref PubMed Scopus (27) Google Scholar, 9Benesch R. Benesch R.E. Edalji R. Kwong S. Biochem. Biophys. Res. Commun. 1978; 81: 1307-1312Crossref PubMed Scopus (17) Google Scholar, 10Benesch R.E. Kwong S. Edalji R. Benesch R. J. Biol. Chem. 1979; 254: 8169-8172Abstract Full Text PDF PubMed Google Scholar, 11Benesch R.E. Kwong S. Benesch R. Nature. 1982; 299: 231-234Crossref PubMed Scopus (30) Google Scholar, 12Nagel R.L. Bookchin R.M. Johnson J. Labie D. Wajcman H. Isaac-Sodeye W.A. Honing G.R. Schiliro G. Crookston J.H. Matsutomo K. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 670-672Crossref PubMed Scopus (175) Google Scholar, 13Goldberg M.A. Husson M.A. Bunn H.F. J. Biol. Chem. 1977; 252: 3414-3421Abstract Full Text PDF PubMed Google Scholar, 14Nagel R.L. Johnson J. Bookchin R.M. Garel M.C. Rosa J. Schiliro G. Wajcman H. Labie D. Moo-Penn W. Castro O. Nature. 1980; 283: 832-834Crossref PubMed Scopus (71) Google Scholar, 15Eaton W.A. Hofrichter J. Adv. Protein Chem. 1990; 40: 63-279Crossref PubMed Scopus (525) Google Scholar, 16Dumoulin A. Padovan J.C. Manning L.R. Popowicz A. Winslow R.M. Chait B.T. Manning J.M. J. Biol. Chem. 1998; 273: 35032-35038Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 17Himanen J.P. Mirza U.A. Chait B.T. Bookchin R.M. Manning J.M. J. Biol. Chem. 1996; 271: 25152-25156Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 18Himanen J.P. Popowicz A.M. Manning J.M. Blood. 1997; 89: 4196-4203Crossref PubMed Google Scholar, 19Li X. Himanen J.P. Martin de Llano J.J. Padovan J.C. Chait B.T. Manning J.M. Biotechnol. Appl. Biochem. 1999; 29: 165-184PubMed Google Scholar, 20Li X. Briehl R.W. Bookchin R.M. Josephs R. Wei B. Manning J.M. Ferrone F.A. J. Biol. Chem. 2002; 277: 13479-13487Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 21Manning J.M. Dumoulin A. Li X. Manning L.R. J. Biol. Chem. 1998; 273: 19359-19362Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22Nacharaju P. Roy R.P. White S.P. Nagel R.L. Acharya A.S. J. Biol. Chem. 1997; 272: 27869-27876Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 23Roy R.P. Acharya A.S. Methods Enzymol. 1994; 231: 194-215Crossref PubMed Scopus (18) Google Scholar, 24Sivaram M.V. Sudha R. Roy R.P. J. Biol. Chem. 2001; 276: 18209-18215Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), and electron microscopic studies (25Dykes G.W. Crepeau R.H. Edelstein S.J. J. Mol. Biol. 1979; 130: 451-472Crossref PubMed Scopus (151) Google Scholar) have led to a 14-stranded model of the fiber (26Cretegny I. Edelstein S.J. J. Mol. Biol. 1993; 230: 733-738Crossref PubMed Scopus (53) Google Scholar, 27Watowich S.J. Gross L.J. Josephs R. J. Struct. Biol. 1997; 111: 161-179Crossref Scopus (47) Google Scholar). These strands appear as seven double strands of the type found in HbS crystals, albeit with a slight twist caused by fiber packing. The models specify several amino acid residues from both α- and β-chains that participate in inter- or intra-double-strand contacts stabilizing the fiber structure. By and large, the fiber models agree with the solution polymerization experiments of mutant hemoglobins in that polymerization-sensitive mutations are usually located in fiber contact regions (27Watowich S.J. Gross L.J. Josephs R. J. Struct. Biol. 1997; 111: 161-179Crossref Scopus (47) Google Scholar). However, the models present an approximate description of the HbS fiber that are limited to the identity of the contact residues and totally lack information on the hierarchy of interactions or additive or synergistic effect of two or more contact residues (4Harrington D.J. Adachi K. Royer Jr., W.E. J. Mol. Biol. 1997; 272: 398-407Crossref PubMed Scopus (132) Google Scholar, 26Cretegny I. Edelstein S.J. J. Mol. Biol. 1993; 230: 733-738Crossref PubMed Scopus (53) Google Scholar, 27Watowich S.J. Gross L.J. Josephs R. J. Struct. Biol. 1997; 111: 161-179Crossref Scopus (47) Google Scholar). Furthermore, the models project a static view of the contact sites and do not provide any clues about the influence of flexibility or dynamics of a given contact region on fiber assembly. Here we have examined the consequence of flexibility changes at the boundary of the A- and B-helix (AB region) of the α-chain on the polymerization of HbS. The linkage of interaction in the polymerization process, between the AB region and a remote site located at the EF corner of the α-chain, has also been explored. The AB region of the α-chain contains several contact sites of the fiber (α16, α20, and α23) and lies in spatial proximity to a cluster of GH corner residues that are implicated in fiber interactions (residues α113-α116). Besides, AB-GH corners of the α-chain engage in extensive inter-tetrameric interactions with AB-GH corners of the β-chain, yielding physiologically important intra-double-strand axial contacts of the fiber. Therefore, it is conceivable that even subtle alterations of the conformational dynamics generated through rational amino acid changes at non-contact sites at the AB or GH corners of α/β chains might influence the HbS polymerization by perturbing the inter-tetrameric interaction interface of the fiber. We have tested this surmise by replacing two Ala residues of the AB corner of the α-chain, Ala19 (AB1) and Ala21 (B2), with the relatively more flexible Gly residues or less flexible α-aminoisobutyric acid (Aib) residues (Fig. 1A). Aib (also called α-methylalanine), an analog of alanine, is a non-protein amino acid found in microbial peptides that form membrane channels. Aib exhibits higher helix-forming ability compared with Ala residues, which has the highest helical propensity among the 20 coded amino acids. Model studies on short peptides have shown that incorporation of one or more Aib residues significantly enhance the helical backbone structure (28Karle I.L. Acc. Chem. Res. 1999; 32: 693-701Crossref Scopus (58) Google Scholar, 29Balaram P. Curr. Opin. Struct. Biol. 1992; 2: 845-851Crossref Scopus (203) Google Scholar). The stabilizing effect of Aib emanates from the presence of an extra methyl group (relative to Ala) that restricts the peptide backbone torsional angles (ϕ, Ψ) to helical conformation. In fact, Gly → Ala substitutions also introduce a methyl group on Cα atom, and similar effects, albeit to a lesser extent than Aib, are realized due to Ala. Therefore, a hierarchy of conformational constraint is expected to be generated at the AB corner while traversing from Gly to Ala to Aib; the ensuing sequence of residues 18–22 at the AB corner (Fig. 1B) with Gly substitution (GGHGG) is likely to be potentially more flexible than the native sequence (GAHAG), which in turn, would be more flexible than the sequence generated by Aib replacements (GAibHAibG). We have earlier successfully employed a semi-synthetic reaction, namely, V8 protease-mediated coupling of α1–30 and α31–141 fragments to produce α141 (α-globin), for the construction of mutant α-subunits (23Roy R.P. Acharya A.S. Methods Enzymol. 1994; 231: 194-215Crossref PubMed Scopus (18) Google Scholar, 24Sivaram M.V. Sudha R. Roy R.P. J. Biol. Chem. 2001; 276: 18209-18215Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). This strategy, although restrictive in nature, is especially suited for the incorporation of non-coded Aib residues and, in appropriate situations, could be effectively utilized for the introduction of 20 standard amino acids. Accordingly, we have constructed the aforementioned α-globin mutants containing Gly or Aib residues at 19 and 21 positions through the α-globin semisynthetic reaction. We have also taken advantage of the fortuitous co-presence of Gly residues at the 19/21 positions and a contact point mutation at position 78 (EF7, Asn → His or Gln) in the α-chain of the Hanuman langur (Presbytus entellus) hemoglobin (30Matsuda G. Maita T. Nakashima Y. Barnabas J. Ranjekar P.K. Gandhi N.S. Int. J. Pept. Protein Res. 1973; 5: 423-425Crossref PubMed Scopus (20) Google Scholar) to authenticate the role of conformational flexibility of the AB region as well as to explore linkage of interactions, if any, between these two remote regions of the tetramer. The ion-exchange resins (CM52 and DE52) were obtained from Whatman. The chemicals used in peptide synthesis were obtained from Nova Biochem. The hemoglobins from homozygous (SS) sickle cell patients and Hanuman langur (HL) were purified from their red cell lysates by DE52 and CM52 chromatography according to standard procedures. Heme-bound individual subunits of hemoglobins were obtained as hydroxymercuribenzoate derivatives following the procedure described by Bucci (31Bucci E. Methods Enzymol. 1981; 76: 97-106Crossref PubMed Scopus (49) Google Scholar). The heme-free chains were obtained by acidacetone precipitation. Isolation and Characterization of HL α-Chain—Hemoglobin from the HL erythrocyte lysate was subjected to CM52 cation-exchange chromatography, and the elution profile was monitored at 540 nm. The chromatographic profile revealed the presence of two hemoglobin components that were present in the ratio of 45:55. This finding was unexpected in view of the fact that HL is known to contain a single hemoglobin. To locate the “variant” chain(s), the two hemoglobins (referred to as HL1 and HL2 in the order of elution) from CM52 chromatography were subjected to RP-HPLC for separation of their α and β globins. The RP-HPLC profile of the above HL hemoglobin samples (HL1 and HL2) displayed two globin peaks with 1:1 stoichiometry. The globin peaks from respective hemoglobins were collected and subjected to positive ion electro-spray mass spectrometry (ESMS). The experimental mass of the first peak in each case (15,896.44 Da and 15,896.39 Da, respectively) was similar and in agreement with the reported sequence (calculated mass, 15,895.24 Da) of langur β-globin. In contrast, the mass of the second peak of RP-HPLC, corresponding to the α-chain from the two samples, was different. Interestingly, the mass of this chain from HL2, 15,123.03 Da, fitted well to the calculated mass (15,121.36 Da) of the documented sequence of the HL α-chain (30Matsuda G. Maita T. Nakashima Y. Barnabas J. Ranjekar P.K. Gandhi N.S. Int. J. Pept. Protein Res. 1973; 5: 423-425Crossref PubMed Scopus (20) Google Scholar) that differs from human at only three sites (Gly19, Gly21, His78). The experimental mass of the α-chain from the HL1 sample was, however, less by about 10 Da (15,113.81). Thus the heterogeneity in the HL hemoglobin was localized in the α-chain. To define the region of sequence change(s), the two α-chains of HL were subjected to tryptic peptide mapping (Fig. 2). A comparison of the peptide maps of the two HL chains revealed altered retention times for two peptides namely, T8+9 (residues 61–90) and T9 (residues 62–90), suggesting that the origin of sequence variation in the two chains was the region encompassing residues 61–90. Further, the positions of the three peptides, T4 (17Himanen J.P. Mirza U.A. Chait B.T. Bookchin R.M. Manning J.M. J. Biol. Chem. 1996; 271: 25152-25156Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 18Himanen J.P. Popowicz A.M. Manning J.M. Blood. 1997; 89: 4196-4203Crossref PubMed Google Scholar, 19Li X. Himanen J.P. Martin de Llano J.J. Padovan J.C. Chait B.T. Manning J.M. Biotechnol. Appl. Biochem. 1999; 29: 165-184PubMed Google Scholar, 20Li X. Briehl R.W. Bookchin R.M. Josephs R. Wei B. Manning J.M. Ferrone F.A. J. Biol. Chem. 2002; 277: 13479-13487Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 21Manning J.M. Dumoulin A. Li X. Manning L.R. J. Biol. Chem. 1998; 273: 19359-19362Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22Nacharaju P. Roy R.P. White S.P. Nagel R.L. Acharya A.S. J. Biol. Chem. 1997; 272: 27869-27876Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 23Roy R.P. Acharya A.S. Methods Enzymol. 1994; 231: 194-215Crossref PubMed Scopus (18) Google Scholar, 24Sivaram M.V. Sudha R. Roy R.P. J. Biol. Chem. 2001; 276: 18209-18215Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 25Dykes G.W. Crepeau R.H. Edelstein S.J. J. Mol. Biol. 1979; 130: 451-472Crossref PubMed Scopus (151) Google Scholar, 26Cretegny I. Edelstein S.J. J. Mol. Biol. 1993; 230: 733-738Crossref PubMed Scopus (53) Google Scholar, 27Watowich S.J. Gross L.J. Josephs R. J. Struct. Biol. 1997; 111: 161-179Crossref Scopus (47) Google Scholar, 28Karle I.L. Acc. Chem. Res. 1999; 32: 693-701Crossref Scopus (58) Google Scholar, 29Balaram P. Curr. Opin. Struct. Biol. 1992; 2: 845-851Crossref Scopus (203) Google Scholar, 30Matsuda G. Maita T. Nakashima Y. Barnabas J. Ranjekar P.K. Gandhi N.S. Int. J. Pept. Protein Res. 1973; 5: 423-425Crossref PubMed Scopus (20) Google Scholar, 31Bucci E. Methods Enzymol. 1981; 76: 97-106Crossref PubMed Scopus (49) Google Scholar), T8+9, and T9 were distinct between the human and the two HL chains. This result is consistent with the fact that the HL α-chain sequence is known to differ from human at positions 19, 21, and 78. For locating exactly the site of sequence changes in the variant chain, T8+9 and T9 peptide fragments were isolated by RP-HPLC and subjected to 25 cycles of Edman degradation. The sequence assignment of 22 and 25 residues in T9 and T8+9, respectively, were made unambiguously and were found to be identical to the human sequence, except at the 78th position. Although the 17th and 18th cycles of amino acid sequencing of T9 and T8+9 (corresponding to the 78th position of the α-chain) peptides derived from HL1 α-chain yielded a Gln residue, the same peptides of HL2 α-chain yielded His. The sequencing results were further confirmed by mass measurements. T8+9 fragment obtained from human sequence had a mass of 3124.89 Da, which was expected for the presence of Asn at 78th position (calculated mass, 3125.51). In the case of HL1α and HL2α, the masses of the T8+9 fragments were found to be 3139.22 and 3148.94 Da, respectively. These values fit the Asn → Gln or His substitutions in T8+9 fragment and are also in agreement with the mass of the corresponding intact α-chains. Thus, the presence of two kinds of α-chain in HL hemoglobin was unequivocally established. Although these chains show three sequence changes relative to the human α-chain (Gly19, Gly21, and His78/Gln78), they differ from each other only at a single residue at position 78 (His or Gln). Semi-synthesis of Mutant α-Chains—The complementary fragments of α-globin, α1–30 and α31–141, were obtained by subjecting the V8 protease digest of α-globin to Sephadex G50 chromatography (24Sivaram M.V. Sudha R. Roy R.P. J. Biol. Chem. 2001; 276: 18209-18215Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The sequence comprising α1–30 with Gly or Aib residues at positions 19 and 21 was assembled by solid-phase peptide synthesis. The respective purified peptides were ligated with the complementary fragment (α31–141) derived from human or langur chains by V8 protease-mediated ligation reaction (23Roy R.P. Acharya A.S. Methods Enzymol. 1994; 231: 194-215Crossref PubMed Scopus (18) Google Scholar, 24Sivaram M.V. Sudha R. Roy R.P. J. Biol. Chem. 2001; 276: 18209-18215Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Semi-synthetic mutant globins were purified by CM52 chromatography in the presence of 8 m urea as described previously (24Sivaram M.V. Sudha R. Roy R.P. J. Biol. Chem. 2001; 276: 18209-18215Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). ESMS, tryptic peptide mapping, and amino acid sequencing were employed to establish the chemical identity of the mutant chains. Generation of Mutant HbS Tetramers—The heme-free mutant globins were reconstituted with βs-chain and heme to tetrameric mutant HbS through the “alloplex” route (32Yip Y.K. Waks M. Beychok S. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 64-68Crossref PubMed Scopus (61) Google Scholar) and according to the protocol described by Sivaram et al. (24Sivaram M.V. Sudha R. Roy R.P. J. Biol. Chem. 2001; 276: 18209-18215Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). HbS tetramers composed of heme-bound HL α-chain were prepared by incubating the α-chain (0.5 mm) with 1.2-fold molar excess of βs-chain (50 mm Tris-HCl, pH 7.4, containing 1mm dithiothreitol and 1 mm EDTA) at 4 °C for 24 h. The tetramers were obtained in pure form by CM52 chromatography. The purity was further established by analytical anion-exchange chromatography on a Mono Q HR 5/5 column using FPLC (AKTA, Amersham Biosciences). Circular Dichroism (CD) Spectroscopy—CD spectra of HbS constructs were recorded on a J710 spectropolarimeter using a 1-mm path-length cell. The respective buffer baselines were subtracted from the sample data, and the final spectrum was represented as mean residue ellipticity in degree/cm2/dmol units. Oxygen-affinity Measurements—Oxygen-affinity measurements were done on a Hemox analyzer (TCS Medical Products, New Hope, PA) in Bis-Tris buffer, pH 7.4. The value of partial oxygen pressure at 50% saturation (P50) and Hill coefficient (nH) was calculated from each dissociation curve. Measurements of Gelation Concentration (Csat)—The Csat was measured in the presence of 12 g/dl concentration of dextran (70 kDa) as described by Bookchin et al. (33Bookchin R.M. Balaz T. Wang Z. Joseph R. Lew V.L. J. Biol. Chem. 1999; 274: 6689-6697Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). This quasi-physiological method has been amply demonstrated to produce the same information as that of standard Csat measurements but at a much-reduced hemoglobin concentration. HbS samples were taken in 50 mm phosphate buffer (pH 7.5) and were allowed to polymerize in the presence of sodium dithionite at 37 °C. The concentration of HbS in soluble phase (Csat) was determined after centrifugation of the gelation mixture at room temperature. Molecular Dynamics (MD) Simulation—MD simulations on the α-chain of HbS and the mutants were carried out using the GROMACS suite of programs (34Lindahl E. Hess B. van der Spoel D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar). The initial structure of the native protein was taken from the high-resolution x-ray crystal structure (4Harrington D.J. Adachi K. Royer Jr., W.E. J. Mol. Biol. 1997; 272: 398-407Crossref PubMed Scopus (132) Google Scholar) of HbS (Protein Data Bank code 2HBS). The structures of the mutants were generated interactively using Insight II (Accelrys). The initial model structures were placed in a simulation box of size 42.8 × 31.5 × 42.7 Å3. The closest distance from any protein atom to the walls of the box was not less than 9 Å. The system was then solvated by adding a bath of simple point charge (35Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Hermans J. Pullman B. Intermolecular Forces. Reidel, Dordrecht, The Netherlands1981: 331-342Google Scholar) waters in such a way that the density of the system was as close to 1 as possible. The α-chain of HbS including the heme has an equal number of negative and positive charges; hence, there was no need of adding counterions for ensuring charge neutrality. The system was first relaxed by energy minimization using the steepest descent algorithm (1000 steps). Subsequently, the added solvent around the protein was allowed to equilibrate by means of position-restrained MD for 25 ps and energy was minimized again with steepest descents (1000 steps). Constant-volume MD simulations were carried out for 2 ns at 300 K with periodic boundary conditions imposed. The bond lengths and bond angles of the protein were restrained around their equilibrium values. Rationale and Design of Study—The hemoglobin molecules in the crystal pack as two strands of HbS molecules. Hemoglobin tetramers in each strand are connected through a network of axial contacts (4Harrington D.J. Adachi K. Royer Jr., W.E. J. Mol. Biol. 1997; 272: 398-407Crossref PubMed Scopus (132) Google Scholar). These contacts involve AB and GH regions of both the α- and β-chains that include αLys16, αHis20, αPro114, αAla115, αGlu116, βGly16, βLys17, βGlu22, βHis117, βPhe118, βGly119, and βGlu121. 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. We chose sites Ala19 and Ala21 at the AB region of the α-chain to address the above issue based on the fact that these are not contact points themselves, but their replacements with Gly residues generate a sequence (18GGHGG22) that has the propensity to enhance the conformational flexibility of the AB region. Secondly, the readily available V8 protease-mediated α-globin semisynthetic procedure permits us to incorporate non-coded Aib residues that are expected to contrast the effects because of Gly residues and confer conformational rigidity to the AB region. Molecular Dynamics Simulations of the Mutant α-Chains—We carried out MD simulations studies on native (Ala) and mutant (Gly or Aib) α-chains to investigate the extent of dynamic perturbations in the mutant chains. The MD simulations for each mutant α-chain were checked for stability by monitoring several global properties like the radius of gyration, root mean squared deviation from the initial structure as well as the kinetic and potential energies. These parameters were found to stabilize after about 0.1 ns and, hence, data from 0.1–2 ns were used for all subsequent analyses. The root-mean-squared fluctuations (RMSF) of the Cα carbon atoms were calculated for the native (αA19-A21) and mutant chains (αA19G/A21G and αA19Aib/A21Aib) and is shown in Fig. 3A. The crystallographic temperature factors for the starting structure are also shown for comparison (Fig. 3B). Structural fluctuations in the mutant α-chains are, overall, similar to those of the native chain and follow the same trend as that seen in the experimental structure, suggesting that the introduction of mutations did not cause any large-scale deviation of the dynamical properties of the α-chain. The regions around residues 42 and 93 within the CD and FG corner, respectively, show the most fluctuations and a large difference between the native α-chain and its mutants. Both of these regions are particularly close to the highly mobile propionate side chains of the heme moiety. The RMSF difference in these regions also varies when different time windows of the simulation were used for calculation. This result indicates that, perhaps because of strong interaction with the highly mobile propionate side chains, the dynamics in these regions becomes much more complicated, and it was not possible to sample fully the total conformational space even during 2 ns of MD simulation, resulting in the observed difference in conformational fluctuation between the native α-chain and its mutants. The RMSF values in the AB corner regions rise to a local maximum in all three trajectories (Ala, Gly, Aib), indicating that this region is relatively mobile. Introduction of Ala → Gly and Ala → Aib mutations do cause small differences in RMSF values in this region. The GH corner region also lies in a local maximum of the RMSF plot. Although the average fluctuation in the GH region is comparable between the three trajectories, a significant difference between the fluctuation profile of the AB and the GH region is observed. This is clearly apparent in Fig. 4, where the frequency distribution of Cα-Cα distance between pairs of AB and GH corner residues is shown. The distribution of Cα-Cα distance between Ala13 and Glu116 is more variable in the Gly mutant and tends toward larger values. A similar trend is also apparent in the Cα-Cα distance distribution calculated for the Ala13-Leu113 pair. In the case of the Lys16-Glu116 and Lys16-Leu113 pairs, the effect is less pronounced for the Cα-Cα distances, but is clearly visible when one considers the side chain atoms NZ of Lys16 and CD of Glu116 (data not shown). We used dynamical cross-correlation maps to identify correlated movements of residues from MD simulati" @default.
• W2022251609 created "2016-06-24" @default.
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• W2022251609 creator A5039569441 @default.
• W2022251609 creator A5040709715 @default.
• W2022251609 creator A5045269893 @default.
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• W2022251609 date "2004-05-01" @default.
• W2022251609 modified "2023-09-27" @default.
• W2022251609 title "Linkage of Interactions in Sickle Hemoglobin Fiber Assembly" @default.
• W2022251609 cites W1475771111 @default.
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• W2022251609 cites W1693240140 @default.
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• W2022251609 cites W1969553017 @default.
• W2022251609 cites W1974617804 @default.
• W2022251609 cites W1987037884 @default.
• W2022251609 cites W1988630889 @default.
• W2022251609 cites W1997708818 @default.
• W2022251609 cites W2002821156 @default.
• W2022251609 cites W2019134707 @default.
• W2022251609 cites W2019999354 @default.
• W2022251609 cites W2022193577 @default.
• W2022251609 cites W2028226298 @default.
• W2022251609 cites W2044998117 @default.
• W2022251609 cites W2059852102 @default.
• W2022251609 cites W2066275610 @default.
• W2022251609 cites W2073176444 @default.
• W2022251609 cites W2076441433 @default.
• W2022251609 cites W2076837063 @default.
• W2022251609 cites W2079356179 @default.
• W2022251609 cites W2084892616 @default.
• W2022251609 cites W2089819802 @default.
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• W2022251609 cites W2123768693 @default.
• W2022251609 cites W2151285771 @default.
• W2022251609 cites W2154403100 @default.
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• W2022251609 cites W4234960494 @default.
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