TRANSPORT-RELATED CONFORMATIONAL STATES OF THE BAND-3 PROTEIN - PROBING WITH 1-FLUORO-2,4-DINITROBENZENE

The present article provides experimental evidence for previous claims , that Lys 539, without being directly involved in anion binding or tr anslocation, is allosterically linked to the anion binding sites of th e band 3 protein and to some other, as yet unidentified amino acid res idue. The evidence is based on a detailed study of the kinetics of inh ibition of sulphate equilibrium exchange by 1-fluoro-2,4-dinitrobenzen e (N(2)ph-F). It is shown that the mutation of Lys 558 in mouse band 3 , which is homologous to Lys 539 in human band 3, renders the transpor t protein insusceptible to inhibition by N(2)ph-F, confirming that it is the modification of this residue which results in the inhibition of band 3-mediated transport. The investigation of the kinetics of the m odification of human band 3 revealed that the modification is not prec eded by non-covalent N(2)ph-F binding and hence governed by the struct ure of the native protein near Lys 539. In chloride-containing media, the rate constant of dinitrophenylation of Lys 539 is about 15 times h igher than in sulphate-containing media. This suggests that the chemic al nature of the anion species bound to band 3 determines whether Lys 539 exists in a buried or exposed state and hence represents a reporte r group which characterizes the functional state of the transport prot ein. The parameter values describing the effects of anion binding on t he interactions between Lys 539 and an allosterically linked, unidenti fied amino acid residue were determined by means of a mathematical mod el which permitted the quantitative evaluation of the data.