Differences in crystal properties and ligand affinities of an antifluorescyl Fab (4-4-20) in two solvent systems.
An antigen-binding fragment (Fab) from a murine monoclonal antibody (4-4-20) with high affinity for fluorescein was cocrystallized with ligand in polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) in forms suitable for X-ray analyses. In MPD the affinity of the intact antibody for fluorescein was 300 times lower than the value (3.4 x 10(10) M-1) obtained in aqueous buffers. This decreased affinity was manifested by the partial release of bound fluorescein when MPD was added to solutions of liganded Fab during crystallization trials. In PEG, the ligand remained firmly bound to the protein. The liganded Fab crystallized in the monoclinic space group P2(1) in PEG, with a = 58.6, b = 97.2, c = 44.5 A and beta = 95.2 degrees. In MPD the space group was triclinic P1, with a = 58.3, b = 43.4, c = 42.3 A, alpha = 83.9 degrees, beta = 87.6 degrees, and gamma = 84.5 degrees. X-ray diffraction data were collected for both forms to 2.5-A resolution. Surprisingly, the triclinic form of the liganed antifluorescyl Fab had the same space group, closely similar cell dimensions, and practically the same orientation in the unit cell as an unliganded Fab (BV04-01) with activity against single-stranded DNA.
Gibson
A L
AL
Department of Biology, University of Utah, Salt Lake City 84112.
Herron
J N
JN
He
X M
XM
Patrick
V A
VA
Mason
M L
ML
Lin
J N
JN
Kranz
D M
DM
Voss
E W
EW
Edmundson
A B
AB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D003461:Crystallography; D019793:Fluorescein; D005452:Fluoresceins; D005454:Fluorescence Polarization; D006018:Glycols; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000032:analysis*; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D011092:Polyethylene Glycols; D012997:Solvents
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D006018:Glycols; D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D012997:Solvents; D011092:Polyethylene Glycols; C011480:hexylene glycol; D019793:Fluorescein