Nuclear Magnetic Resonance Studies of Immunoglobulins: I. Structure-Function Relationships in Phosphorylcholine-Binding Mouse Myeloma Antibodies. II. ¹⁹F NMR Studies of Trifluoroacetonylated Immunogloblins as a Probe of Antibody Conformation

Chapter 1 This chapter presents a general introduction to mouse myeloma proteins, antibody structure, and the nature of antibody-antigen interactions. The contributions of light and heavy chains to antibody binding properties are also discussed and the 3-dimensional structure of the McPC 603 binding site is examined in some detail. Chapter 2 The interaction of phosphorylcholine-binding mouse myeloma protein McPC 603 and the isotopically substituted hapten phosphorylmethyl-13Ccholine has been investigated using 13C and 31P nuclear magnetic resonance spectroscopy. Upon binding to antibody, upfield shifts of 0.7 and 1.5 ppm are observed for the hapten 13C and 31P resonances, respectively, and both spectra are in the "slow" exchange limit. Linewidth analysis indicates some immobilization of the phosphate group but essentially unrestricted methyl group rotation for the bound hapten. Hapten-antibody dissociation rate constants of 10 and 38 s-1 are calculated from 13c and 31P NMR spectra, respectively, suggesting the possibility of differential dissociation rates for the two opposing ends of the phosphorylcholine molecule. The NMR data are entirely consistent with the known X-ray structure of the McPC 603 Fab'-phosphorylcholine complex . Chapter 3 The binding site interactions between the phosphorylcholine-binding mouse myeloma proteins TEPC 15, W3207, McPC 603, MOPC 167, and MOPC 511 and the isotopically subsituted hapten phosphorylmethyl-13Ccholine have been investigated using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy. Each protein exhibits a unique NMR pattern, but extensive similarities in chemical shift parameters upon binding of hapten to immunoglobulin suggest a significant degree of conservation of important hapten-binding site interactions. Moreover, independent binding studies, in conjunction with the NMR data, allow construction of a simple model of the binding sites of these antibodies, analyzed in terms of the relative strength of interaction between hapten and two main subsites. The NMR evidence supports the view that the heavy chains of these proteins dominate in interacting with bound phosphorylcholine. Chapter 4 We have determined the pH dependencies of the binding affinities of the mouse myeloma immunoglobulins M603, W3207, T15 and M167 for the haptens phosphorylcholine (PC) and L-α-glycerophosphorylcholine (GPC). These affinities are generally maximal near neutral pH with the exception of the binding of PC by Ml67 which is strongest at pH 5.5. These data have helped to clarify the nature and relative importance of the ionic interactions between hapten and antibody. 31P nuclear magnetic resonance (NMR) techniques were used to probe the influence of pH on the microenvironment of the phosphate group of several haptens when these were bound to M603, W3207, T15, M167 and M511. The phosphate subsites of M603, W3207 and Tl5 are electropositive and also show other similarities; those of M167 and M511 have more electronegative character than PC experiences in solution. The two hydrogen bonds known to be formed between M603 and the phosphate oxygens of PC are also involved in binding GPC and are essentially unaffected by pH in the region 3-9. Studies with the hapten 3-trimethyl- amino-1-propanolphosphate show that the binding cavity of M1 67 is substantially wider than those of M603, W3207 and T15. These results lead to a detailed, molecular model of the pH dependent binding of PC and related haptens to these antibodies; they further indicate the roles of various amino acid residues in defining the differing ligand specificities of these antibodies. Chapter 5 The phosphorylcholine (PC) affinities of several hybrid immunoglobulins, formed from recombination of light and heavy chains from parent molecules with PC specificity, have been determined. The results indicate a highly specific light-heavy chain interaction in these antibodies since high PC binding affinities are obtained only with autologous recombinants or with heterologous recombinants in which both parent molecules have a highly similar light chain. It is proposed that the heavy chain residues of these immunoglobulins determine both the primary PC specificity and the differing fine specificities; the important role of the different light chains is then to stabilize unique heavy chain conformations. Chapter 6 The domain structure of antibodies, as well as the localization of various effector functions to specific antibody regions, are discussed. Evidence, from the literature, for the induction of conformational changes in the antibody molecule as a result of antigen binding is presented and the possible nature of such changes is discussed. Chapter 7 The six interchain disulfide bonds of TEPC 15 were trifluoroacetonylated and 19F NMR studies used to probe the magnetic environments of the trifluoroacetonyl reporter groups. The use of various enzymatic fragments of the intact immunoglobulin enabled the observed fluorine signals to be partially assigned to the known locations of the disulfide bonds on the antibody molecule and the distinct chemical environments seen for the 19F labels are in good agreement with the known 3-dimensional structures of antibodies. No change in environment of the trifluoroacetonyl groups was observed upon binding of TEPC 15 to phosphorylcholine or to phosphorylcholine conjugated to a protein carrier. Similar studies with trifluoroacetonylated MOPC 315 binding to 2,4-dinitrophenyl-sensitized sheep red blood cells likewise resulted in no observable changes in the 19F NMR spectrum.