Temperature and pH dependence of the self-association of human spectrin

Gb, Ralston

doi:10.1021/bi00231a011

Cited by 30 publications

(26 citation statements)

References 23 publications

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“…The K d values shown in Table 1 indicate that, in comparison with the native Arg28/Arg45 peptide, Arg45Thr exhibited about a 7-fold reduction in its affinity with Sp␤, whereas Arg45Ser and Arg28Ser exhibited about a 50-to 60-fold reduction in their affinity with Sp␤. The value for Arg28/Arg45 (Table 1) is qualitatively similar to those reported previously, in the range of approximately 10 Ϫ6 to 10 Ϫ7 M. 6,23,24 Using a solid-state assay, we previously obtained the concentration for 50% inhibition (IC 50 ) of 0.14 M for binding of Arg28/Arg45 to Sp␤ 20 and 0.3 M for Arg28/Arg45 and intact ␤-spectrin. 18 Thus, the values in Table 1 are about 3 to 7 times higher than those obtained by a solid-state assay.…”

Section: Association Of Arg28ser Arg45ser and Arg45thr With Sp␤supporting

confidence: 85%

Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin

Park

Johnson

Fung

2002

Blood

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Many spectrin mutations that destabilize tetramer formation and lead to hereditary hemolytic anemias are located at the Nterminal region of ␣-spectrin, with the Arg28 position considered to be a mutation hot spot. We have introduced mutations at positions 28 and 45 into a model peptide, Sp␣1-156, consisting of the first 156 residues in the N-terminal region of ␣-spectrin (␣N). The association of these ␣-spectrin peptides that have single amino acid replacements with a ␤-spectrin model peptide, consisting of the Cterminal region of ␤-spectrin (␤C), was determined, and structural changes due to amino acid replacements were monitored by nuclear magnetic resonance (NMR). We found evidence for similar and very localized structural changes in Sp␣1-156Arg45Thr and Sp␣1-156Arg45Ser, although these 2 mutant peptides associated with ␤-spectrin peptide with significantly differing affinities. The Sp␣1-156Arg28Ser peptide showed an affinity for the ␤-spectrin peptide comparable to that of Sp␣1-156Arg45Ser, but it exhibited substantial and widespread spectral changes. Our results suggest that both Arg45 replacements induce only minor structural perturbations in the first helix of Sp␣1-156, but the Arg28Ser replacement affects both the first helix and the following structural domain. Our results also indicate that the mechanism for reduced spectrin tetramerization is through mutation-induced changes in molecular recognition at the ␣␤-tetramerization site, rather than through conformational disruption, as has been suggested in prior literature. IntroductionThe ␣ and ␤ subunits of human erythrocyte spectrin both consist of multiple homologous sequence motifs, with each motif presumably folding into 3 helices, which bundle to form a structural domain similar to the structures determined for Drosophila spectrin 1 and chicken brain spectrin. 2,3 Both ␣-and ␤-spectrin associate at the N-terminal end of the ␤-subunit and the C-terminal end of the ␣-subunit (dimer nucleation site) with high affinity (nM dissociation constant [K d ] values) to give ␣␤ heterodimers. 4,5 It has been suggested that spectrin dimers then associate to form spectrin tetramers, with an association site at the other end of the dimers, involving 2 sets of identical, low-affinity (M K d ) interactions between the N-terminal region of the ␣-subunit (␣N) of one ␣␤ dimer and the C-terminal region of the ␤-subunit (␤C) in another ␣␤ dimer to give an (␣␤) 2 tetramer. 6,7 Sequence homology studies predict that about 30 residues in this ␣N region, prior to the first structural domain, fold into a helical conformation; likewise, about 60 residues in the ␤C region, following the last structural domain, are predicted to fold into 2 helices. 8,9 These one-and 2-helix regions are termed partial domains for ␣-and ␤-spectrin, respectively. It is assumed that the dimer to tetramer formation involves association of these partial domains to form a triple helical bundle similar to the structural domains. 7,8 Spectrin tetramers in the human erythrocyte play a critical role in ma...

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Section: Association Of Arg28ser Arg45ser and Arg45thr With Sp␤supporting

confidence: 85%

Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin

Park

Johnson

Fung

2002

Blood

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“…The interactions observed in this study appeared to be not affected by ionic strength, implying that a balance of hydrophobic and electrostatic forces may be at work. Such behavior has been seen in interactions of certain other synthetic peptides of ␣-helical coiled coils (44). However, the intact dimer to tetramer equilibrium shows a different behavior; the association weakens with increasing ionic strength (48,49).…”

Section: Discussionmentioning

confidence: 77%

“…Again, the properties of the dimer to tetramer association of intact spectrin differed. For intact dimer to tetramer association over this temperature range, ultracentrifugation studies show that the association was weakened only slightly upon increasing temperature, with K d ϳ0.5 M at 20°C and ϳ1.7 M at 35°C (44). At lower temperatures (e.g.…”

Section: Discussionmentioning

confidence: 87%

“…The association of ␣-and ␤-spectrin at the tetramerization site has been studied with intact spectrin (22,23,44,45) as well as with spectrin fragments (21,23,30,43). It has been shown that a proteolytically produced fragment, ␣I (21), and ␣-recombinant peptides consisting of residues 1-639 (recombinant ␣I) or 1-158 bind ␤-spectrin, whereas peptides consisting of residues 31-639 or 28 -158 do not bind ␤-spectrin (43).…”

Section: Discussionmentioning

confidence: 99%

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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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“…The octameric species was predominant at acidic pH values, whereas the dimeric species was predominant at pH values higher than 7.0. This behavior is infrequent, with most proteins behaving in the opposite way, i.e., the association of protomers in multimers is impaired at acidic pH values (Ralston 1991, Zeng et al 1997, Calvete et al 1999, Hochgrebe et al 2000, Madern et al 2000, Madern et al 2001, Wah et al 2001, Lew et al 2003.…”

Section: Discussionmentioning

confidence: 99%

Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature

d’Abusco

Casadio

Tasco

et al. 2005

Archaea

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SummaryThe recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60 -95°C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques.At pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non-polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.

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Temperature and pH dependence of the self-association of human spectrin

Cited by 30 publications

References 23 publications

Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin

Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin

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

Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature

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