The therapeutically anti-prion active antibody-fragment scFv-W226: paramagnetic relaxation-enhanced NMR spectroscopy aided structure elucidation of the paratope-epitope interface.
Antibodies have become indispensable reagents with numerous applications in biological and biotechnical analysis, in diagnostics as well as in therapy. In all cases, selective interaction with an epitope is crucial and depends on the conformation of the paratope. While epitopes are routinely mapped at high throughput, methods revealing structural insights on a rather short timescale are rare. We here demonstrate paramagnetic relaxation-enhanced (PRE) NMR spectroscopy to be a powerful tool unraveling structural information about epitope-orientation in a groove spanned by the complementary determining regions. In particular, we utilize the spin label TOAC, which is fused to the peptidic epitope using standard solid-phase chemistry and which is characterized by a reduced mobility compared to, e.g., spin labels attached to the side-chain functionalities of cysteine or lysine residues. We apply the method to determine the orientation of helix 1 of the prion protein, which is the epitope for the therapeutically anti-prion active scF(v) fragment W226.
Mangels
Christian
C
Department of Biopolymers, Universitat Bayreuth, Universitatsstrasse 30, 95440 Bayreuth, Germany.
Kellner
Ruth
R
Einsiedel
Jürgen
J
Weiglmeier
Philipp R
PR
Rosch
Paul
P
Gmeiner
Peter
P
Schwarzinger
Stephan
S
D000595:Amino Acid Sequence; D001666:Binding Sites, Antibody; D000939:Epitopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry* / Q000276:immunology; D011487:Protein Conformation; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D013113:Spin Labels
D000939:Epitopes; D011328:Prions; D057127:Single-Chain Antibodies; D013113:Spin Labels