Binding constants for a physiologic electron-transfer protein complex between methylamine dehydrogenase and amicyanin. Effects of ionic strength and bound copper on binding.
Two soluble proteins, methylamine dehydrogenase and amicyanin, form a physiologically relevant complex in which intermolecular electron transfer occurs. To characterize and quantitate the binding of these two weakly-associating proteins, an ultrafiltration binding assay has been developed which involves brief centrifugation of mixtures of proteins in centrifuge concentrators followed by quantitation of proteins on each side of the filtration membrane by HPLC. Under low ionic strength conditions which are optimal for the redox reaction between these proteins, a Kd of 4.5 microM was measured for the methylamine dehydrogenase-amicyanin complex. The Kd increased by 8-fold in the presence of added salt. Apoamicyanin, which is known from crystallographic analysis to be structurally very similar to amicyanin, exhibited a much higher Kd and much less specific binding than did the holoprotein. Apoamicyanin also exhibited apparent self-association at low ionic strength which was not observed with amicyanin. These observations are correlated with the known crystal structures of these proteins, free and in complex, and with the available biochemical information on the interactions of these two proteins.
Davidson
V L
VL
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Graichen
M E
ME
Jones
L H
LH
D001059:Apoproteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D004579:Electron Transport; D008956:Models, Chemical; D009994:Osmolar Concentration; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010231:Paracoccus denitrificans; D011485:Protein Binding; D014462:Ultrafiltration
D001059:Apoproteins; D001426:Bacterial Proteins; C017079:mauC protein, Methylobacterium extorquens; D003300:Copper; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors