Physical‐chemical studies of spectrin

Gb, Ralston

doi:10.1002/jss.400080313

Cited by 50 publications

(26 citation statements)

References 25 publications

(34 reference statements)

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“…An initial series of ITC experiments using wild-type ␣0-1 and ␤16-17 yielded a K d of 8.7 M at 37°C and 1.3 M at 30°C, which are very similar to binding affinities measured using intact spectrin dimers. 12,13,58,59 The values obtained by ITC also agree well with previously determined K d values for the ␣I domain peptide and ␤ monomer obtained in prior HPLC gel filtration assays (7.1 M at 37°C and 3.8 M at 30°C). 15 Furthermore, the binding affinity of the wild-type recombinants, as measured by ITC at 23°C in this study, was similar to binding affinities of ␣0-1 and ␤16-17 measured at 25°C using surface plasmon resonance.…”

Section: He and Hpp ␣-Spectrin Tetramer Site Mutations 5715 Blood 15supporting

confidence: 88%

“…[9][10][11] Two spectrin heterodimers self-associate in a head-to-head orientation to form tetramers, the predominant form of the molecule on red cell membranes. Tetramer assembly, which normally involves 2 head-to-head ␣-␤ associations per tetramer, is a moderate affinity, temperature-dependent association, 12,13 involving small regions near the N-terminus of the ␣ subunit and near the C-terminus of the ␤ subunit that have been hypothesized to form a hybrid 3 helix bundle repeat similar to the typical spectrin type repeat. [14][15][16] Specifically, this hybrid repeat consists of a C helix contributed by the ␣0 partial repeat and an A and B helix contributed by the partial ␤17 repeat (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

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Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site

Gaetani

Mootien

Harper

et al. 2008

Blood

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The most common hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) mutations are ␣-spectrin missense mutations in the dimer-tetramer self-association site. In this study, we systematically compared structural and functional properties of the 14 known HE/HPP mutations located in the ␣-spectrin tetramer binding site. All mutant ␣-spectrin recombinant peptides were well folded, stable structures, with only the R34W mutant exhibiting a slight structural destabilization. In contrast, binding affinities measured by isothermal titration calorimetry were greatly variable, ranging from no detectable binding observed for I24S, R28C, R28H, R28S, and R45S to approximately wild-type binding for R34W and K48R. Binding affinities for the other 7 mutants were reduced by approximately 10-to 100-fold relative to wild-type binding. Some sites, such as R28, were hot spots that were very sensitive to even relatively conservative substitutions, whereas other sites were only moderately perturbed by nonconservative substitutions. The R34W and K48R mutations were particularly intriguing mutations that apparently either destabilize tetramers through mechanisms not probed by the univalent tetramer binding assay or represent polymorphisms rather than the pathogenic mutations responsible for observed clinical symptoms. All ␣0 HE/HPP mutations studied here appear to exert their destabilizing effects through molecular recognition rather than structural mechanisms. IntroductionThe hereditary elliptocytosis (HE) syndromes are a common group of disorders characterized by elliptical erythrocytes on peripheral blood smear. [1][2][3][4] These disorders are characterized by marked clinical, biochemical, and genetic heterogeneity. Most patients with typical HE are asymptomatic, but others have chronic hemolysis or the related disorder hereditary pyropoikilocytosis (HPP). Biochemical and genetic heterogeneity is related to qualitative and/or quantitative defects in one of several erythrocyte membrane skeleton proteins, particularly ␣-spectrin, ␤-spectrin, protein 4.1R, or glycophorin C, which leads to mechanical weakness or fragility of the erythrocyte membrane skeleton.The majority of HE-associated defects occur in spectrin, the principal structural component of the red cell membrane skeleton. Spectrin is a flexible, rope-like molecule formed by antiparallel lateral association of 2 subunits, ␣-and ␤-spectrin, which are primarily composed of many tandem, homologous 106 amino acid motifs, or "spectrin type repeats." [5][6][7] Spectrin repeats are highly stable, independently folding 3 helix bundle units that are responsible for imparting much of the strength and flexibility to the erythrocyte membrane skeleton. For example, when conformational changes of membrane skeleton components are probed in intact red cells using cysteine-specific reagents, spectrin is the only membrane skeleton component showing stress-related increases in labeling indicative of tensile stress-related unfolding of specific domains. 8 Assembly of spectrin he...

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Section: He and Hpp ␣-Spectrin Tetramer Site Mutations 5715 Blood 15supporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site

Gaetani

Mootien

Harper

et al. 2008

Blood

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“…These sequence motifs are suggested to fold into triple ␣-helical bundles (structural domains) with the first and third helices parallel and the intervening second helix antiparallel (14). This proposed structure is supported by recent x-ray (15) and NMR (16) studies of various recombinant spectrin fragments, as well as by spectroscopic studies of intact spectrin (17)(18)(19). The three helices show a pronounced amphipathic character, and the resulting triple-␣-helical bundle structure is thought to be stabilized by the sequestration of the hydrophobic faces of each helix to the interior of the bundle.…”

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confidence: 68%

Interactions of the α-Spectrin N-terminal Region with β-Spectrin

Cherry

Menhart

Fung

1999

Journal of Biological Chemistry

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Spectrin of the erythrocyte membrane skeleton is composed of ␣-and ␤-spectrin, which associate to form heterodimers and tetramers. It has been suggested that a fractional domain (helix C) in the amino-terminal region of ␣-spectrin (N␣ region) bundles with another fractional domain in the carboxyl-terminal region of ␤-spectrin (C␤ region) to yield a triple ␣-helical bundle and that this helical bundling is largely responsible for tetramer formation. However, there are certain objections to assigning a preeminent role to this helical bundling in the tetramerization reactions. We prepared several recombinant peptides of ␣-spectrin fragments spanning only the N␣ region (lacking the dimer nucleation site) and quantitatively studied their interaction with ␤-spectrin. We found that a majority of the interactions were localized, as expected, in the N␣-helix C region but that there was also some contribution from the nonhomologous region. More importantly, the temperature and ionic strength dependence of this interaction in our model peptides was different from that in intact spectrin. We suggest that, although the regions involving the putative helical bundling in ␣-and ␤-spectrin undoubtedly play a significant role in tetramerization, regions distal to the N␣-helix C region in spectrin are also involved in tetramer formation. Structural flexibility and lateral interactions may play a role in spectrin tetramerization.Spectrin is the major component of the erythrocyte membrane skeleton and is thought to be largely responsible for the red blood cell's unique flexibility and deformability (1). Spectrin is composed of two subunits, ␣-spectrin (280 kDa) and ␤-spectrin (246 kDa), which associate to form ␣␤ heterodimers (2, 3), which then associate to form the biologically relevant (␣␤) 2 tetramers and higher order oligomers (4 -6). The tetramer exists as a flexible rodlike molecule that is anchored to the erythrocyte membrane by interactions with certain membrane proteins, most importantly band 3 and band 4.1 (7). This spectrin network imparts mechanical stability to erythrocytes. Erythrocyte membranes in patients suffering from hereditary spherocytosis and hereditary elliptocytosis exhibit unusually high mechanical fragility. A large subset of these diseases are known to be the result of defects in spectrin (8 -11).Amino acid (12) and cDNA (13) sequence analyses show that both ␣-and ␤-spectrin are largely composed of multiple homologous motifs of about 106 amino acid residues. These sequence motifs are suggested to fold into triple ␣-helical bundles (structural domains) with the first and third helices parallel and the intervening second helix antiparallel (14). This proposed structure is supported by recent x-ray (15) and NMR (16) studies of various recombinant spectrin fragments, as well as by spectroscopic studies of intact spectrin (17-19). The three helices show a pronounced amphipathic character, and the resulting triple-␣-helical bundle structure is thought to be stabilized by the sequestration of the hydrophobic face...

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“…Circular dichroism (CD) (12,13) and Fourier transform infrared (FTIR) 1 (14) studies of intact spectrin corroborate this model, indicating that spectrin consists of a large proportion of ␣-helix. However, little experimental information is available on the molecular structure of intact spectrin, in part due to its large size and its structural flexibility which makes nuclear magnetic resonance or x-ray studies difficult.…”

mentioning

confidence: 79%

Peptides with More than One 106-amino Acid Sequence Motif Are Needed to Mimic the Structural Stability of Spectrin

Menhart

Mitchell²,

Lusitani³

et al. 1996

Journal of Biological Chemistry

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The primary sequence of human erythrocyte spectrin contains repetitive homologous sequence motifs of approximately 106 amino acids with 22 such motifs in the ␣-subunit and 17 in the ␤-subunit. These homologous sequence motifs have been proposed to form domains with a triple-helical bundle type structure (Speicher, In this study, we show that these sequence motifs, while they do form compact proteolytically resistant units, are not completely independent. Peptides composed of two or three such motifs in tandem are substantially more stable than peptides composed of a single motif, as measured by proteolysis or by fluorescence or circular dichroism studies of urea or thermal denaturation. Circular dichroism and infrared spectroscopy measurements also indicate that these larger, more stable peptides exhibit greater secondary structure. In these respects, the peptides with tandem sequence motifs are more similar to intact spectrin than the peptide with a single sequence motif. Thus, we conclude that peptides with more than one sequence motif model spectrin more adequately than the peptides with one sequence motif, and that these sequence motifs are not completely independent domains.DHuman erythrocytes contain a dense, two-dimensional network of spectrin and other proteins that provides support to the lipid bilayer and maintains erythrocyte deformability (1). Spectrin, comprising ␣-and ␤-subunits, plays a critical role in maintaining the architecture and therefore the integrity of the red cell membrane. Many hereditary hemolytic anemias involve spectrin mutations (2-4). Thus, it is important to understand the structural properties of spectrin. The bulk (about 90%) of the primary structure of spectrin comprises repetitive homologous units of approximately 106 amino acids in length. Several other proteins, including brain spectrin (fodrin), dystrophin, and ␣-actinin, also have similar repetitive amino acid units in their sequences and are known as the spectrin superfamily (5).A triple-helical bundle model has been suggested for the 106-amino acid sequence motif in which the three helices are aligned side by side, with the first and third parallel and the intervening second helix antiparallel (6 -8). X-ray diffraction studies of one such sequence motif unit from a non-erythroid spectrin support this model (9). In these x-ray studies, the peptide used was found to form a homodimer containing two triple-helical structures, in which two of the three helices are contributed by one monomer and the remaining helix by the other monomer. It is thought that this peculiar arrangement may be an artifact of crystallization, and the true structure of a single unit may be similar to the earlier suggested triplehelical bundle with a zigzag arrangement of the helices (9, 10). This arrangement aligns the amino-and carboxyl-terminal residues at opposite ends of this triple-helical bundle, and thus sequential motifs are thought to be linked in tandem in intact spectrin, producing a very long rod-shaped molecule, approximately 100 nm in l...

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Physical‐chemical studies of spectrin

Cited by 50 publications

References 25 publications

Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site

Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site

Interactions of the α-Spectrin N-terminal Region with β-Spectrin

Peptides with More than One 106-amino Acid Sequence Motif Are Needed to Mimic the Structural Stability of Spectrin

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