Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs.
Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs.
复制标题
通过噬菌体展示的 scFv 可以区分红系和非红系 β 血影蛋白四聚化区域的明显结构差异。
DOI：
10.1002/pro.617
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Fung,LW-M
中科院分区：
文献类型：
--
作者：
Song,Yuanli;Antoniou,Chloe;Memic,Adnan;Kay,BrianK;Fung,LW-M
We have screened a human immunoglobulin single‐chain variable fragment (scFv) phage library against the C‐terminal tetramerization regions of erythroid and nonerythroid beta spectrin (βI‐C1 and βII‐C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid β‐spectrin isoforms. We have identified interacting scFvs, with clones “G5” and “A2” binding only to βI‐C1, and clone “F11” binding only to βII‐C1. TheKdvalues, estimated by competitive enzyme‐linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1–0.3 μM. A more quantitativeKdvalue from isothermal titration calorimetry experiments with the recombinant G5 and βI‐C1 was 0.15 μM. The α‐spectrin fragments (model proteins), αI‐N1 and αII‐N1, competed with the βI‐C1, or βII‐C1, binding scFvs, with inhibitory concentration (IC50) values of ∼50 μMfor αI‐N1, and ∼0.5 μMfor αII‐N1. Our predicted structures of βI‐C1 and βII‐C1 suggest that the Helix B′ of the C‐terminal partial domain of βI differs from that of βII. Consequently, an unstructured region downstream of Helix B′ in βI may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in βII was helical, and βII did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C‐termini of different β‐spectrin isoforms to regulate α‐ and β‐spectrin association to form functional spectrin tetramers, and may sort β‐spectrin isoforms to their specific cellular localizations.
