Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs
✍ Scribed by Yuanli Song; Chloe Antoniou; Adnan Memic; Brian K. Kay; L.W.-M. Fung
- Book ID
- 105356626
- Publisher
- Cold Spring Harbor Laboratory Press
- Year
- 2011
- Tongue
- English
- Weight
- 699 KB
- Volume
- 20
- Category
- Article
- ISSN
- 0961-8368
- DOI
- 10.1002/pro.617
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✦ Synopsis
Abstract
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. The K~d~ values, estimated by competitive enzyme‐linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1–0.3 μ__M__. A more quantitative K~d~ value from isothermal titration calorimetry experiments with the recombinant G5 and βI‐C1 was 0.15 μ__M__. The α‐spectrin fragments (model proteins), αI‐N1 and αII‐N1, competed with the βI‐C1, or βII‐C1, binding scFvs, with inhibitory concentration (IC~50~) values of ∼50 μ__M__ for αI‐N1, and ∼0.5 μ__M__ for 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.