Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

Song, Yuanli; Antoniou, Chloe; Memić, Adnan; Kay, Brian K.; Fung, Leslie W.‐M.

doi:10.1002/pro.617

Cited by 4 publications

(3 citation statements)

References 68 publications

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“…Although the amino terminal region of the α subunit and carboxy terminal region of the β subunit of nonerythroid (brain) spectrin exhibit~60 and 70 % similarity with corresponding regions of erythroid spectrin [26,27], the two spectrin isoforms appear different in their structure and function. Crystal structure and phage-displayed single-chain variable fragment analysis show that the tetramerization site or the selfassociating domain of brain spectrin is different from that of erythroid spectrin [28,29]. This is further supported by the observation that the heterodimer of brain spectrin forms tetramer that is~15 times stronger than the corresponding tetramer formed by erythroid spectrin [30].…”

Section: Introductionmentioning

confidence: 61%

Organization and Dynamics of Tryptophan Residues in Brain Spectrin: Novel Insight into Conformational Flexibility

et al. 2015

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Brain spectrin enjoys overall structural and sequence similarity with erythroid spectrin, but less is known about its function. We utilized the fluorescence properties of tryptophan residues to monitor their organization and dynamics in brain spectrin. Keeping in mind the functional relevance of hydrophobic binding sites in brain spectrin, we monitored the organization and dynamics of brain spectrin bound to PRODAN. Results from red edge excitation shift (REES) indicate that the organization of tryptophans in brain spectrin is maintained to a considerable extent even after denaturation. These results are supported by acrylamide quenching experiments. To the best of our knowledge, these results constitute the first report of the presence of residual structure in urea-denatured brain spectrin. We further show from REES and time-resolved emission spectra that PRODAN bound to brain spectrin is characterized by motional restriction. These results provide useful information on the differences between erythroid spectrin and brain spectrin.

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Section: Introductionmentioning

confidence: 61%

Organization and Dynamics of Tryptophan Residues in Brain Spectrin: Novel Insight into Conformational Flexibility

et al. 2015

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“…The purity of H70N was monitored by PAGE gel electrophoresis 11 . The mass of H70N was analyzed by high resolution mass spectrometry (RRC).…”

Section: Protein Analysismentioning

confidence: 99%

Site-Directed Mutagenesis of N5-Carboxyaminoimidazole Ribonucleotide Mutase (Class I PurE) in Bacillus anthracis

Silas

Wolf

Fung

2011

J Undergrad Research UIC

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Anthrax, the infection caused by the Gram-positive pathogen Bacillus anthracis (B. anthracis), is fatal if untreated, and some strains of B. anthracis have been found to be resistant to currently available antibiotics. The development of broad spectrum antibiotics is needed to treat the resistive strains. In antibiotic development, we have targeted B. anthracis Class I PurE enzyme (Ba PurE) as a unique and necessary enzyme in the de novo purine biosynthesis pathway, since the inactivation of this gene prevents B. anthraci growth in human serum, resulting in decreased bacterial proliferation. To identify inhibitors to $Ba$PurE, structural information on the substrate binding to its active site is needed. However, it is difficult to obtain crystals of Ba PurE with the substrate molecule in its binding site since upon binding to PurE, the substrate molecule is converted to the product molecule. An alternative approach is to create mutants of PurE that exhibit no enzymatic activity and do not convert the substrate to product, but still allow the substrate to bind to the active site. Then, the structure of mutant PurE with bound substrate can be obtained. We have identified a histidine residue at position 70 as the target of mutation to give an inactive enzyme. After successfully preparing the recombinant protein H70N, we have found that it exhibited no enzyme activity. This mutant will be useful in future experimentation to identify inhibitors of Ba PurE.

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“…Spectrin tetramerization involves interaction of the lone helix (Helix C’) at the N-terminal region of α-spectrin of one αβ heterodimer and the two helices (Helix A’ and Helix B’) at the C-terminal region of the β-spectrin on another heterodimer [23 - 26]. This interaction involves hydrophobic residue clustering, salt bridges and hydrogen bonds [25 - 29].…”

Section: Introductionmentioning

confidence: 99%

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

Sevinc

Fung

2011

Cellular and Molecular Biology Letters

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With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697-2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IPβII-C s). The proteins include a fragment (residues 38-284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74-442) of septin 8 isoform c, a fragment (residues 704-953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146-614) of zinc-finger protein 251, and a fragment (residues 284-435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) [1] on spectrin tetramer formation. The results showed that 3 IPβII-C s were able to bind βII-C even in the presence of αII-N, and 4 IPαII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.

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Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

Cited by 4 publications

References 68 publications

Organization and Dynamics of Tryptophan Residues in Brain Spectrin: Novel Insight into Conformational Flexibility

Organization and Dynamics of Tryptophan Residues in Brain Spectrin: Novel Insight into Conformational Flexibility

Site-Directed Mutagenesis of N5-Carboxyaminoimidazole Ribonucleotide Mutase (Class I PurE) in Bacillus anthracis

Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

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