INTER-ACTIVE-SITE DISTANCE AND SOLUTION DYNAMICS OF A BIVALENT-BISPECIFIC SINGLE-CHAIN ANTIBODY MOLECULE

The solution dynamics of a bivalent bispecific single-chain antibody ( BiSCA) specific against fluorescein (Fl) and single-stranded DNA (ssDN A) were investigated. Fluorescence resonance energy transfer (FRET) st udies were performed in order to estimate the average distances, R, be tween the anti-Fl and the anti-ssDNA active sites. In separate experim ents, either 2-(dimethylamino)naphthalene-5-sulfonyl chloride coupled to the 5' end of an oligothymidylate polymer of 6 residues length (2,5 -DNS-dT(6)) served as energy donor to Fl or eosin isothiocyanate coupl ed to the 5' end of an oligothymidylate polymer of 6 residues length ( eosin-dT(6)) served as energy acceptor from Fl. Labeling of dT(6) with 2,5-DNS or eosin did not significantly interfere with recognition by the anti-ssDNA binding site. With the 2,5-DNS/Fl energy transfer pair, the calculated values of R(k(2) = 2/3), R(min), and R(max) were 44, 3 7, and 54 Angstrom, respectively. With Fl/eosin (opposite direction of FRET), values of 40, 33, and 51 Angstrom, respectively, were obtained . Considering the sizes of the two SCA domains and the length of the i nterdomain polypeptide linker, an R value of approximately 140 Angstro m would be expected for the extended molecule. The fact that measured R distances were on average 3-fold shorter than 140 Angstrom indicated that BiSCA was not an extended and rigid molecule. The efficiency of energy transfer increased with increasing temperature in the range of 10-30 degrees C, suggesting that conformational fluctuations of the pr otein resulted in decreased average distance between BiSCA active site s. The decay of the intrinsic fluorescence anisotropy in BiSCA was dom inated by local mobility of tryptophan residues, as indicated by the p redominance of a subnanosecond rotational correlation time component. A second rotational correlation time of approximately 7 ns was also fo und. The expected value for the overall rotation of the 57-kDa BiSCA m olecule is approximately 21 ns. Comparison between measured and expect ed rotational correlation times indicated that the fluorescence anisot ropy decay in BiSCA did not report overall tumbling of the whole molec ule but independent rotational motions of the two SCA domains. These r esults, along with FRET data, suggest that BiSCA displays considerable conformational dynamics in solution, which likely results from flexib ility of the linker.