Protein Engineering vol. 8 no. 8 pp. 749-761, 1995
© 1995 Oxford University Press
RESEARCH-ARTICLE |
1.85 Å structure of anti-fluorescein 4-4-20 Fab
Enzon Incorporated (previously Genex Corporation), Research and Development Department 40 Kingsbridge Road, Piscataway, NJ 08854–3998, USA 1Present address: Berlex BioBiosciences, 15049 San Pablo Avenue, PO Box 4099, Richmond, CA 94804, USA 2Present address: Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850, USA 3Present address: Department of Biologies Research, Division of Communicable Disease and Immunology, Building 501 Forest Glen Section, Walter Reed Army Institute of Research, Washington, DC 20307, USA 4Department of Microbiology, University of Illinois Urbana- Champaign, IL 61801, USA 5Present address: Du Pont Merck Pharmaceutical Co., Experimental Station, Building 328, PO Box 80228, Wilmington, DE 19880, USA
1To whom correspondence should be addressed
The crystal complex of fluorescein bound to the high-affinity anti-fluorescein 4-4-20 Fab {Ka = 1010 M–1 at 2°C) has been determined at 1.85 Å. Isomorphous crystals of two isoelectric forms (p1 = 7.5 and 7.9) of the antifluorescein 4-4-20 Fab, an IgG2A [Gibson et al (1988)Proteins: Struct. Funct Genet., 3, 155–160], have been grown. Both complexes crystallize with one molecule in the asymmetric unit in space group P1, with a = 42.75 Å, b =43.87 Å, c = 58.17 Å, 
= 95.15° , ß = 86.85° and 
= 98.01°. The final structure has an R value of 0.188 at 1.85 Å resolution. Interactions between bound fluorescein, the complementarity-determining regions (CDRs) of the Fab and the active-site mutants of the 4-4-20 single-chain Fv will be discussed. Differences were found between the structure reported here and the previously reported 2.7 Å 4-4-20 Fab structure [Herron et al. (1989) Proteins: Struct. Fund., 5, 271–280]. Our structure determination was based on 26 328 unique reflections — four times the amount of data used in the previous report. Differences in the two structures could be explained by differences in interpreting the electron density maps at the various resolutions. The r.m.s. deviations between the variable and constant domains of the two structures were 0.77 and 1.54 Å, respectively. Four regions of the light chain and four regions of the heavy chain had r.m.s. backbone deviations of >4 Å. The most significant of these was the conformation of the light chain CDR 1.
Keywords: anti-fluorescein Fab/complementarity determining/region/crystal complex/structure determination
Received December 9, 1994; revised June 13, 1995; accepted June 20, 1995.
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