Backbone assignment,secondary structure and Protein A binding of an isolated,human antibody VH domain |
| |
Authors: | Lutz Riechmann Julian Davies |
| |
Institution: | (1) MRC Laboratory of Molecular Biology, Hills Road, CB2 2QH Cambridge, U.K. |
| |
Abstract: | Summary Antibody heavy chain variable domains (VH) lacking their light chain domain (VL) partner are prime candidates for the design of minimum-size immunoreagents. To obtain structural information about isolated VH domains, a human VH was labelled with 15N or doubly labelled with both 15N and 13C and was studied by heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the 1H and 15N main-chain signals were assigned through two-dimensional TOCSY and NOESY experiments on the unlabelled VH and three-dimensional heteronuclear multiple quantum correlation TOCSY and NOESY experiments on the 15N-labelled VH. Four short stretches of the polypeptide chain could only be assigned on the basis of three-dimensional HNCA and HN(CO)CA experiments on the 13C-/15N-labelled protein. Long-range interstrand backbone NOEs suggest the presence of two adjacent -sheets formed by altogether nine antiparallel -strands. 3JH
NHC coupling constants and the location of slowly exchanging backbone amides support this interpretation. The secondary structure of the isolated VH is identical to that of heavy chain variable domains in intact antibodies, where VH domains are packed against a VL domain. The backbone assignments of the VH made it possible to locate its Protein A binding site. Chemical shift movements after complexing with the IgG binding fragment of Protein A indicate binding through one of the two -sheets of the VH. This -sheet is solvent exposed in intact antibodies. The Protein A binding site obviously differs from that on the Fc portion of immunoglobulins and is unique to members of the human VHIII gene subgroup.Abbreviations CDR
complementarity determining region
- CHAPS
(cholamidopropyl)-dimethylammonio]-1-propanesulfonate
- DQF-COSY
double-quantum-filtered correlated spectroscopy
- Fab
antigen binding antibody fragment
- Fc
crystallisable antibody fragment
- Fv
heterodimer of VH and VL
- H1 (2, 3)
hypervariable loop 1 (2, 3)
- IgG
immunoglobulin G
- NOE
nuclear Overhauser effect
- NOESY
nuclear Overhauser enhancement spectroscopy
- HMQC
heteronuclear multiple quantum correlation spectroscopy
- HSQC
heteronuclear single quantum correlation spectroscopy
- scFv
single chain Fv
- TOCSY
total correlation spectroscopy
- TPPI
time-proportional phase incrementation
- VH
antibody heavy chain variable region
- VL
antibody light chain variable region. Mutants are denoted by the wild-type amino acid (one-letter code), follwed by the residue number and the new amino acid |
| |
Keywords: | Human immunoglobulin Camel Detergent Protein A Heteronuclear NMR Secondary structure |
本文献已被 SpringerLink 等数据库收录! |
|