A combined proton and phosphorus-31 nuclear magnetic resonance investigation of the combining site of M603, a phosphocholine-binding myeloma protein.
Phosphorus-31 nuclear magnetic resonance (NMR) studies on the two phosphorus nuclei of the phosphonium analogue (Me3P+CH2CH2OPO3(2-)) of phosphocholine are used to monitor the charged subsites in the phosphocholine-binding immunoglobulin A mouse myeloma M603. Comparison of the 270-MHz 1H NMR difference spectrum on addition of either this analogue or phosphocholine to M603 and the almost identical changes in the pKa values of the phosphate groups on binding to M603 confirm that the analogue is a good model for phosphocholine. The pKa of the phosphate groups is decreased by 0.5 unit on binding to M603, which is consistent with the phosphate group being hydrogen bonding to Tyr-33H and Arg-95L, as suggested from the X-ray structure, and also implies that the binding energies for the mono- and dianion are similar. The P+Me3 moiety is used to probe the electrostatic interactions in the choline subsite. Titration of the chemical shift of the phosphonium phosphorus reflects a group on the protein that has a pKa value of less than or equal to 5, which from the refined X-ray structure (D.R. Davies, personal communication) of the site is assigned to Asp-97L. The choline subsite is monitored by using 1H NMR difference spectra, which indicates that the subsite is highly aromatic as expected from the crystal structure that places Trp-107H and Tyr-100L in this subsite. The ring current interactions from these rings can account for the 1H NMR chemical shift data on choline.
D001665:Binding Sites; D002794:Choline / Q000031:analogs & derivatives*; D007070:Immunoglobulin A / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments; D007136:Immunoglobulins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009194:Myeloma Proteins / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons
D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments; D007136:Immunoglobulins; D009194:Myeloma Proteins; D010761:Phosphorus Radioisotopes; D011522:Protons; C017551:myeloma immunoglobulins; D010767:Phosphorylcholine; D002794:Choline