Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

Adachi, K.; Kim, J.Y.; Konitzer, P; Asakura, Toshio; Saviola, B.; Surrey, Saul

doi:10.1016/0014-5793(93)81130-r

Cited by 27 publications

(29 citation statements)

References 36 publications

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“…␤112 Ser, ␤112 Val, ␤112 Thr, and ␤112 Asp) were constructed and expressed using the pHE2␤ plasmid vector that contains cDNAs coding for each ␤ chain variant and methionine aminopeptidase which was originally developed to express ␣ and ␤ chains at the same time (8,9). The basic strategy for generation of these variants by site-specific mutagenesis of the normal ␤ chain involves recombination/polymerase chain reaction as described previously (10). Clones were subjected to DNA sequence analysis of the entire ␤-globin cDNA region using site-specific primers and fluorescently tagged terminators in a cycle sequencing reaction in which extension products were analyzed on an automated DNA sequencer.…”

Section: Expression Of Soluble Recombinant Human ␤-Globin Chain Variantsmentioning

confidence: 99%

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Yamaguchi¹,

Pang²,

Reddy³

et al. 1998

Journal of Biological Chemistry

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In order to assess the role of ␤112 Cys in homo-and hetero-tetrameric hemoglobin formation, we expressed four ␤112 variants (␤ 112Cys3 Asp , ␤ 112Cys3 Ser , ␤ 112Cys3 Thr , and ␤ 112Cys3 Val ) and studied assembly with ␣ chains in vitro. ␤112 Cys is normally present at ␤ 1 ␤ 2 and ␣ 1 ␤ 1 interaction sites in homo-(␤ 4 ) and hetero-tetramers (␣ 2 ␤ 2 ). ␤ 4 formation in vitro was influenced by the amino acid at ␤112. ␤112 Asp completely inhibited formation of homo-tetramers, whereas ␤112 Ser showed only slight inhibition. In contrast, ␤112 Thr or Val enhanced homotetramer formation compared with ␤ A chains. Association constants for homo-tetramer formation increased in the order of ␤ 112Cys3 Ser , ␤ A , ␤ 112Cys3 Thr , and ␤ 112Cys3 Val , whereas the value for ␤ 112Cys3 Asp was zero under the same conditions. These ␤112 changes also affected in vitro ␣ 2 ␤ 2 hetero-tetramer formation. Order of ␣ 2 ␤ 2 formation under limiting ␣-globin chain conditions showed Hb ␤C112S > Hb A > Hb S ‫؍‬ Hb ␤C112T ‫؍‬ Hb ␤C112V > > > Hb ␤C112D. Hb ␤112D can form tetrameric hemoglobin, but this ␤112 change promotes dissociation into ␣ and ␤ chains instead of ␣␤ dimer formation upon dilution. These results indicate that amino acids at ␣ 1 ␤ 1 interaction sites such as ␤112 on the G helix play a key role in stable ␣␤ dimer formation. Our findings suggest, in addition to electrostatic interaction between ␣ and ␤ chains, that dissociation of ␤ 4 homo-tetramers to monomers and hydrophobic interactions of the ␤112 amino acid with ␣ chains governs stable ␣ 1 ␤ 1 interactions, which then results in formation of functional hemoglobin tetramers. Information gained from these studies should increase our understanding of the mechanism of assembly of multi-subunit proteins.Equimolar amounts of ␣-and ␤-globin chains of human hemoglobin self assemble to form ␣ 2 ␤ 2 tetramer. In addition, isolated ␤ chains also assemble to form ␤ 4 homo-tetramers (1). Extensive previous studies using naturally occurring variants and our recent studies using recombinant ␤ chain variants showed that affinity between ␣ and ␤ chains is promoted by negatively charged ␤ chains and is independent of charge location on the surface except at the ␣ 1 ␤ 1 interaction site (2-5). Our previous studies showed that affinity is promoted by negatively charged ␤ chains up to a maximum of two additional net negative charges (5). In addition, we showed that ␤112 Cys located at an ␣ 1 ␤ 1 interaction site on the G helix is critical for facilitating formation of stable ␣␤ dimers which then form functional hemoglobin tetramers. We also demonstrated that ␤ 112Cys3 Asp inhibits formation of stable ␣ 1 ␤ 1 and ␤ 1 ␤ 2 interactions in ␣ 2 ␤ 2 and ␤ 4 tetramers, respectively (5).X-ray diffraction analysis of ␤ 4 and ␣ 2 ␤ 2 showed that ␤112 Cys on the G helix is involved in ␤ 1 ␤ 2 and ␣ 1 ␤ 1 subunit interactions, respectively, and that there is similarity between the quaternary structure of carbonmonoxy ␤ 4 and carbonmonoxy ␣ 2 ␤ 2 tetramers (6). The detailed role of ␣ 1 ␤ 1 interaction s...

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Section: Expression Of Soluble Recombinant Human ␤-Globin Chain Variantsmentioning

confidence: 99%

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Yamaguchi¹,

Pang²,

Reddy³

et al. 1998

Journal of Biological Chemistry

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“…A hydrophobic amino acid on the surface of the tetramer, such as Ile at codon 4, might significantly increase surface hydrophobicity. In fact, site-directed mutagenesis studies show decreased stability to mechanical agitation as ␤6 amino acid hydrophobicity increases (43). The Thr 3 Ile change at codon 4 in the ␦-chain significantly decreases mechanical stability of this variant, whereas heat stability was not affected, similar to Hb S. Hb A 2 has high affinity for red cell membranes (44).…”

Section: Expression Of ␦-Chain Structural Variants and Functional Chamentioning

confidence: 98%

“…Once the different mutations were constructed in pGS188␦ (see below), insertion of the corresponding XhoI fragments into pGS389 was then repeated, resulting in construction of wild type and variant Hb A 2 expression vectors. Homologous PCR recombination was used for mutagenesis (25). The initial wild type ␦-globin cDNA amplified by RT-PCR contained an unanticipated PCR-induced T 3 C at codon 141 Leu 3 Pro that was identical to one of the ␦-chain variants.…”

Section: Aagtgcccttgaggttgtccmentioning

confidence: 99%

“…Yeast growth, harvesting, and purification of Hb A 2 and variants were performed as described previously (25). Purified Hb A 2 variants were subjected to electrospray mass analysis (Fisons Instruments, VG Biotech, Altricham, UK) using the multiply charged ion peaks from the ␣-globin chain (molecular mass, 15,126.4 Da) as an external reference for mass scale calibrations (28).…”

Section: Aagtgcccttgaggttgtccmentioning

confidence: 99%

“…The C 3 T at codon 4 Thr 3 Ile, G 3 T at codon 27 Ala 3 Ser, C 3 T at codon 116 Arg 3 Cys, and T 3 C at codon 141 Leu 3 Pro were introduced separately into normal ␦-globin cDNA using homologous PCR recombination (25). Variant ␦-globin chains were expressed in a yeast system, which results in production of soluble Hb A 2 tetramers, which can be readily isolated.…”

Section: Expression Of ␦-Chain Structural Variants and Functional Chamentioning

confidence: 99%

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Expression Studies of δ-Globin Gene Alleles Associated with Reduced Hemoglobin A2 Levels in Greek Cypriots

Trifillis¹,

Adachi

Yamaguchi

et al. 1996

Journal of Biological Chemistry

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We previously identified five ␦-globin gene alleles associated with reduced hemoglobin (Hb) A 2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298 -3305). We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing. In addition, variant Hb A 2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G 3 T change at codon 27 and the A 3 G change in IVS-2 both affect RNA splicing, whereas the C 3 T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A 2 variants expressed in yeast were similar to that of wild type Hb A 2 . None of the three variant Hb A 2 tetramers (Thr 3 Ile at codon 4 (Hb ␦T4I), Ala 3 Ser at codon 27 (Hb ␦A27S), and Arg 3 Cys at codon 116 (Hb ␦R116C)) showed decreased heat stability compared with Hb A 2 , whereas the Hb ␦T4I variant showed highest instability to mechanical agitation. Coexpression in yeast of ␣-globin chain and the ␦-chain variant containing a Leu 3 Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability. These studies show the probable cause for decreased Hb A 2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of ␦-chains, which inhibits assembly with ␣-chains, and/or reduced assembly is suggested for the other three alleles.

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Clinical and haematological characteristics of 38 individuals with Hb G‐Makassar in Malaysia

Esa,

Mohamad,

Hamzah

et al. 2023

eJHaem

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Haemoglobin (Hb) G‐Makassar is a rare Hb variant. It presents a diagnostic challenge as it imitates sickle Hb (Hb S) in standard electrophoresis and high‐performance liquid chromatography assays requiring DNA analysis to confirm diagnosis. Both have point mutations in codon 6, exon 1 in the β‐globin (HBB) gene with different pathogenicities. This study describes the clinical phenotype, haematology and genotype of Hb G‐Makassar. Clinical and laboratory data of 38 cases of Hb G‐Makassar over 8 years were analysed. Hb G‐Makassar was confirmed by a direct sequencing of HBB gene and co‐inheritance of α‐thalassaemia determined through multiplex gap‐PCR and multiplex Amplification Refractory Mutation System polymerase chain reaction. All cases were Malays, predominantly from Terengganu (n = 20, 52.6%). There were 14 (36.8%) males and 24 (63.2%) females with median age of 25 years. Majority (n = 33, 86.8%) had features of thalassaemia trait with mean ± SD for Hb, mean cell volume (MCV) and mean cell haemoglobin (MCH) as 13.21 g/dL ± 1.69, 73.06 ± 4.48 fL and 24.71 ± 1.82 pg, respectively. None had evidence of haemolysis or thromboembolic complications. Six genotypes were identified; ßG‐Makassar/ß,αα/αα (n = 19, 50.0%), ßG‐Makassar/ßE,αα/αα (n = 4, 10.5%), ßG‐Makassar/ßNewYork,αα/αα (n = 1, 2.6%), ßG‐Makassar/ß,αα/‐α (n = 11, 28.9%), ßG‐Makassar/ß,αα/αAdanaα (n = 2, 5.3%) and ßG‐Makassar/ß,αα/–SEA (n = 1, 2.6%). The ßG‐Makassar/ß,αα/αα showed that features of thalassaemia trait with mean ± SD for Hb, MCV and MCH were 13.74 g/dL ± 2.40, 76.18 ± 6.02 fL and 25.79 ± 2.41 pg, respectively. This is the largest study reporting a significant number of Hb G‐Makassar in Malaysia. Although the mutation is similar to Hb S, the phenotype is benign.

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Effects of β6 amino acid hydrophobicity on stability and solubility of hemoglobin tetramers

Cited by 27 publications

References 36 publications

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

Expression Studies of δ-Globin Gene Alleles Associated with Reduced Hemoglobin A2 Levels in Greek Cypriots

Clinical and haematological characteristics of 38 individuals with Hb G‐Makassar in Malaysia

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