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

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 (?I-C1 and ?II-C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid ?-spectrin isoforms. We have identified interacting scFvs, with clones "G5" and "A2" binding only to ?I-C1, and clone "F11" binding only to ?II-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 ?M. A more quantitative K(d) value from isothermal titration calorimetry experiments with the recombinant G5 and ?I-C1 was 0.15 ?M. The ?-spectrin fragments (model proteins), ?I-N1 and ?II-N1, competed with the ?I-C1, or ?II-C1, binding scFvs, with inhibitory concentration (IC(50) ) values of ?50 ?M for ?I-N1, and ?0.5 ?M for ?II-N1. Our predicted structures of ?I-C1 and ?II-C1 suggest that the Helix B' of the C-terminal partial domain of ?I differs from that of ?II. Consequently, an unstructured region downstream of Helix B' in ?I may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in ?II was helical, and ?II did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C-termini of different ?-spectrin isoforms to regulate ?- and ?-spectrin association to form functional spectrin tetramers, and may sort ?-spectrin isoforms to their specific cellular localizations.

SUBMITTER: Song Y 

PROVIDER: S-EPMC3125871 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature