Human serum transferrin N-lobe (hTF/2N) contains three conserved tryptophan residues, Trp8, Trp128 and Trp264, located in three different environments. The present report addresses the different contributions of the three tryptophan residues to the UV–visible, fluorescence and NMR spectra of hTF/2N and the effect of the mutations at each tryptophan residue on the iron-binding properties of the protein. Trp8 resides in a hydrophobic box containing a cluster of three phenylalanine side chains and is H bonded through the indole N to an adjacent water cluster lying between two β-sheets containing Trp8 and Lys296 respectively. The fluorescence of Trp8 may be quenched by the benzene rings. The apparent increase in the rate of iron release from the Trp8→Tyr mutant could be due to the interference of the mutation with the H-bond linkage resulting in an effect on the second shell network. The partial quenching in the fluorescence of Trp128 results from the nearby His119 residue. Difference-fluorescence spectra reveal that any protein containing Trp128 shows a blue shift upon binding metal ion, and the NMR signal of Trp128 broadens out and disappears upon the binding of paramagnetic metals to the protein. These data imply that Trp128 is a major fluorescent and NMR reporter group for metal binding, and possibly for cleft closure in hTF/2N. Trp264 is located on the surface of the protein and does not connect to any functional residues. This explains the facts that Trp264 is the major contributor to both the absorbance and fluorescence spectra, has a strong NMR signal and the mutation at Trp264 has little effect on the iron-binding and release behaviours of the protein.
Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe
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Qing-Yu HE, Anne B. MASON, Barbara A. LYONS, Beatrice M. TAM, Vinh NGUYEN, Ross T.A. MACGILLIVRAY, Robert C. WOODWORTH; Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe. Biochem J 1 March 2001; 354 (2): 423–429. doi: https://doi.org/10.1042/bj3540423
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