Inter-active-site distance and solution dynamics of a bivalent-bispecific single-chain antibody molecule

Biochemistry. 1994 Aug 23;33(33):10100-8. doi: 10.1021/bi00199a038.


The solution dynamics of a bivalent bispecific single-chain antibody (BiSCA) specific against fluorescein (Fl) and single-stranded DNA (ssDNA) were investigated. Fluorescence resonance energy transfer (FRET) studies were performed in order to estimate the average distances, R, between the anti-Fl and the anti-ssDNA active sites. In separate experiments, either 2-(dimethylamino)naphthalene-5-sulfonyl chloride coupled to the 5' end of an oligothymidylate polymer of 6 residues length (2,5-DNS-dT6) served as energy donor to Fl or eosin isothiocyanate coupled to the 5' end of an oligothymidylate polymer of 6 residues length (eosin-dT6) served as energy acceptor from Fl. Labeling of dT6 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(k2 = 2/3), R(min), and R(max) were 44, 37, and 54 A, respectively. With Fl/eosin (opposite direction of FRET), values of 40, 33, and 51 A, respectively, were obtained. Considering the sizes of the two SCA domains and the length of the interdomain polypeptide linker, an R value of approximately 140 A would be expected for the extended molecule. The fact that measured R distances were on average 3-fold shorter than 140 A 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 protein resulted in decreased average distance between BiSCA active sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antibodies, Bispecific / chemistry*
  • Antigens / immunology
  • Binding Sites, Antibody*
  • DNA, Single-Stranded / immunology
  • Energy Transfer
  • Fluorescein
  • Fluoresceins
  • Fluorescence
  • Fluorescence Polarization
  • Hot Temperature
  • Protein Conformation
  • Solutions


  • Antibodies, Bispecific
  • Antigens
  • DNA, Single-Stranded
  • Fluoresceins
  • Solutions
  • Fluorescein