Complete assignment of the methionyl carbonyl carbon resonances in switch variant anti-dansyl antibodies labeled with [1-13C]methionine.
A 13C NMR study is reported of switch variant anti-dansyl antibodies developed by Dangl et al. [(1982) Cytometry 2, 395-401], who had used the fluorescence-activated cell sorter to select and clone these variants. These switch variant antibodies possess the identical VH, VL, and CL domains in conjunction with different heavy chain constant regions. In the present study, switch variant antibodies of IgG1, IgG2a, and IgG2b subclasses were used along with a short-chain IgG2a antibody, in which the entire CH1 domain is deleted. The switch variant antibodies were specifically labeled with [1-13C]methionine by growing hybridoma cells in serum-free medium. Assignments of all the methionyl carbonyl carbon resonances have been completed by using the intact antibodies along with their fragments and recombined proteins in which either heavy or light chain is labeled. A double labeling method [Kainosho, M., & Tsuji, T. (1982) Biochemistry 21, 6273-6279] has played a crucial role in the process of the spectral assignments. The strategy used for the assignments has been described in detail. In incorporating 15N-labeled amino acids into the antibodies for the double labeling, isotope dilution caused a serious problem except in the cases of [alpha-15N]lysine and [15N]threonine, both of which cannot become the substrate of transaminases. It was found that beta-chloro-L-alanine is most effective in suppressing the isotope scrambling. So far, spectral assignments by the double labeling method have been possible with 15N-labeled Ala, His, Ile, Lys, Met, Ser, Thr, Tyr, and Val.(ABSTRACT TRUNCATED AT 250 WORDS)
Kato
K
K
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Matsunaga
C
C
Igarashi
T
T
Kim
H
H
Odaka
A
A
Shimada
I
I
Arata
Y
Y
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002460:Cell Line; D003619:Dansyl Compounds; D014644:Genetic Variation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D007553:Isotope Labeling / Q000379:methods; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine; D008961:Models, Structural; D009587:Nitrogen Isotopes; D011487:Protein Conformation
D002247:Carbon Isotopes; D003619:Dansyl Compounds; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D008715:Methionine