Magnetic resonance of a monoclonal anti-spin-label antibody

Anglister, Jacob; Frey, Tom; McConnell, Harden M.

doi:10.1021/bi00301a016

Cited by 68 publications

(45 citation statements)

References 17 publications

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“…A complication in this type of distance measurement arises if the spin-label adopts more than one conformation relative to the protein (3). An electron paramagnetic resonance spectrum of AN02 with the spinlabel hapten shows the spin-label to be tumbling at the same rate as would be expected for AN02 (2). The distances from the spin-label of seven AN02 tyrosines were measured by varying the amount of spin-label in the binding site (4).…”

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confidence: 88%

“…A simple NMR difference spectrum, antibody alone minus antibody with bound spin-label, is dominated by resonances from protons that are near the electron spin. Anglister et al (2) used this effect to gain information about the amino acid composition of the binding site of AN02, a monoclonal antibody raised against a spinlabeled dinitrophenyl hapten (DNP-SL). Growth of the AN02-producing cell line in medium containing selected deuterated amino acids results in virtually complete incorporation of these deuterated amino acids into the antibody.…”

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confidence: 99%

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Sequences of 12 monoclonal anti-dinitrophenyl spin-label antibodies for NMR studies.

Leahy

Rule

Whittaker

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Eleven monoclonal antibodies specific for a spin-labeled dinitrophenyl hapten (DNP-SL) have been produced for use in NMR studies. They have been named AN01 and AN03-AN12. The stability constants for the association of these antibodies with DNP-SL and related haptens were measured by fluorescence quenching and ranged from 5.0 x 104 M-1 to >1.0 x 108 M-1. cDNA clones coding for the heavy and light chains of each antibody and of an additional anti-DNP-SL monoclonal antibody, AN02, have been isolated. The nucleic acid sequence of the 5' end of each clone has been determined, and the amino acid sequence of the variable regions of each antibody has been deduced from the cDNA sequence. The sequences are relatively heterogeneous, but both the heavy and the light chains of AN01 and AN03 are derived from the same variable-region gene families as those of the AN02 antibody. AN07 has a heavy chain that is related to that of AN02, and AN09 has a related light chain. AN05 and AN06 are unrelated to AN02 but share virtually identical heavy and light chains. Preliminary NMR difference spectra comparing related antibodies show that sequence-specific assignment of resonances is possible. Such spectra also provide a measure of structural relatedness.Many different magnetic resonance techniques can be used to gain structural information about antibodies in solution (1). The combining sites of antibodies specific for spin-labeled molecules are particularly accessible to study by nuclear magnetic resonance (NMR). The spin-label broadens the NMR signals of nearby (<17 A) protons in a strongly distance-dependent manner. A simple NMR difference spectrum, antibody alone minus antibody with bound spin-label, is dominated by resonances from protons that are near the electron spin. Anglister et al. (2) used this effect to gain information about the amino acid composition of the binding site of AN02, a monoclonal antibody raised against a spinlabeled dinitrophenyl hapten (DNP-SL). Growth of the AN02-producing cell line in medium containing selected deuterated amino acids results in virtually complete incorporation of these deuterated amino acids into the antibody. This selective deuteration permits the assignment of resonances to specific amino acid types. Considerable simplification of spectra is afforded through the use of partially deuterated amino acids to remove splitting ofthe resonances. Measurement of the broadening effect of the spin-label on specific resonance signals at various binding-site occupancies allows the calculation of the distance between these protons and the electron spin. A complication in this type of distance measurement arises if the spin-label adopts more than one conformation relative to the protein (3). An electron paramagnetic resonance spectrum of AN02 with the spinlabel hapten shows the spin-label to be tumbling at the same rate as would be expected for AN02 (2). The distances from the spin-label of seven AN02 tyrosines were measured by varying the amount of spin-label in the binding site (4). Such dis...

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confidence: 88%

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confidence: 99%

Sequences of 12 monoclonal anti-dinitrophenyl spin-label antibodies for NMR studies.

Leahy

Rule

Whittaker

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Independent crystal structures of an antibody both with and without antigen have not yet been solved. We report here the crystallization of the Fab fragment of AN02, a monoclonal antibody specific for a spin-labelled dinitrophenyl hapten, with and without hapten (for the chemical structure of this hapten see Anglister et al,' (1984a)). …”

Section: Introductionmentioning

confidence: 99%

“…Culturing the antibody producing cells in media containing specific deuterated amino acids results in virtually complete incorporation of the deuterated amino acids into the antibody. The spin-label's broadening effect, in combination with deuteration, has enabled identification of NMR signals from amino acids in the hapten binding site and measurement of the distance between many of these residues and the electron spin (Anglister et al, 1984a;1984b;1987).…”

Section: Introductionmentioning

confidence: 99%