Intrinsic fluorescence of the P-glycoprotein multidrug transporter: Sensitivity of tryptophan residues to binding of drugs and nucleotides

P-glycoprotein is a member of the ATP binding cassette family of membrane p roteins, and acts as an ATP-driven efflux pump for a diverse group of hydro phobic drugs, natural products, and peptides, The side chains of aromatic a mino acids have been proposed to play an important role in recognition and binding of substrates by P-glycoprotein. Steady-state and lifetime fluoresc ence techniques were used to probe the environment of the 11 tryptophan res idues within purified functional P-glycoprotein, and their response to bind ing of nucleotides and substrates. The emission spectrum of P-glycoprotein indicated that these residues are present in a relatively nonpolar environm ent, and time-resolved experiments showed the existence of at least two lif etimes. Quenching studies with acrylamide and iodide indicated that those t ryptophan residues predominantly contributing to fluorescence emission are buried within the protein structure. Only small differences in Stern-Volmer quenching constants were noted on binding of nucleotides and drugs, arguin g against large changes in tryptophan accessibility following substrate bin ding. P-glycoprotein fluorescence was highly quenched on binding of fluores cent nucleotides, and moderately quenched by ATP, ADP, and AMP-PNP, suggest ing that the site for nucleotide binding is located relatively close to try ptophan residues. Drugs, modulators, hydrophobic peptides, and nucleotides quenched the fluorescence of P-glycoprotein in a saturable fashion, allowin g estimation of dissociation constants. Many compounds exhibited biphasic q uenching, suggesting the existence of multiple drug binding sites. The quen ching observed for many substrates was attributable largely to resonance en ergy transfer, indicating that these compounds may be located close to tryp tophan residues within, or adjacent to, the membrane-bound domains. Thus, t he regions of P-glycoprotein involved in nucleotide and drug binding appear to be packed together compactly, which would facilitate coupling of ATP hy drolysis to drug transport.