Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe

Human serum transferrin N-lobe (hTF/2N) contains three conserved tryptophan residues, Trp(8), Trp(128) and Trp(264), located in three different enviro nments. The present report addresses the different contributions of the thr ee tryptophan residues to the UV-visible, fluorescence and NMR spectra of h TF/2N and the effect of the mutations at each tryptophan residue on the iro n-binding properties of the protein. Trp(8) resides in a hydrophobic box co ntaining a cluster of three phenylalanine side chains and is H bonded throu gh the indole N to an adjacent water cluster lying between two beta -sheets containing Trp(8) and Lys(296) respectively. The fluorescence of Trp(8) ma y be quenched by the benzene rings. The apparent increase in the rate of ir on release from the Trp(8) --> Tyr mutant could be due to the interference of the mutation with the H-bond linkage resulting in an effect on the secon d shell network. The partial quenching in the fluorescence of Trp(128) resu lts from the nearby His(119) residue. Difference-fluorescence spectra revea l that any protein containing Trp(128) shows a blue shift upon binding meta l ion, and the NMR signal of Trp(128) broadens out and disappears upon the binding of paramagnetic metals to the protein. These data imply that Trp(12 8) is a major fluorescent and NMR reporter group for metal binding, and pos sibly for cleft closure in hTF/2N. Trp(264) is located on the surface of th e protein and does not connect to any functional residues. This explains th e facts that Trp(264) is the major contributor to both the absorbance and f luorescence spectra, has a strong NMR signal and the mutation at Trp(264) h as little effect on the iron-binding and release behaviours of the protein.