UVM Theses and Dissertations
Format:
Print
Author:
James, Nicholas G.
Dept./Program:
Biochemistry
Year:
2009
Degree:
PhD
Abstract:
Human serum transferrin (hTF), a bilobal glycoprotein (80 kDa), is the major transporter of iron in the human body through its ability to reversible bind two ferric ions. The single chain polypeptide folds to form two homologous lobes (N- and C-lobe), each of which provides a suitable environment for a single ferric ion to bind. Spectral kinetic assays provide iron release rate constants, which give insight into the effect a mutation has on iron release in the absence or presence of the specific transferrin receptor. These kinetic assays are based on the unique spectral properties of hTF when iron is bound since coordination of iron produces a ligand to metal charge transfer (LMCT) band centered at 470 nm. Also, the fluorescence emission from hTF is quenched ~ 70% when iron is bound. This attenuation has been described as energy transfer from Trp residues to the LMCT band. Monitoring the recovery of the intrinsic emission over time, under endosomal like conditions (pH ~5.6), has been widely used to derive iron release rate constants due to the extreme sensitivity of fluorescence. This dissertation focuses on the conformational events surrounding iron release and the effects on the Trp fluorescence reporting these events.
Iron release (monitoring the change in fluorescence) from recombinant isolated hTF N-lobe (residues 1-337) is reported by three Trp residues (at positions 8, 128, and 264) in two events (8.9 mid⁻¹ and 1.3 min⁻¹). Direct monitoring of iron loss via decrease in the visible absorption band at 470 nm definitively established that the faster component using intrinsic fluorescence is produced by iron release. Double Trp mutants were created to determine which of the Trp residues contributed to the iron release signal and to define the second event. Iron release from each construct at pH 5.6 revealed that both Trp128 and Trp264 are efficiently quenched when iron is bound. Based on the data from the Trp mutants, the second event was modeled as global conformational relaxation after iron removal reported by Trp128 and Trp264.
Kinetic studies on a monoferric-C-lobe construct revealed that the iron release from this lobe is slow and follows a monoexponential function with a lag ~ 50 sec. Mutation of the Trp residues in the C-lobe (at positions 344, 441, 460 and 550) indicated that the main contributor to the iron release signal is Trp460 and suggested that the lag may be ascribed to lobe-lobe cooperativity. When in the complex with the transferrin receptor, kinetic assays with the C-lobe Trp mutants revealed alterations of the Trp441 and Trp550 fluorescence emission, indicating receptor mediated conformational changes around these residues.
Using multifrequency phase fluorometry the excited-state properties of the individual Trp residues in the hTF N-lobe are reported here for the first time. These experiments were designed to investigate how iron binding affects the lifetimes of the individual Trp residues in the N-lobe and to quantitatively assign FRET efficiencies to each. The R₀ values calculated from the spectral data clearly indicated that FRET is possible. However, Trp264 undergoes dramatic changes in its lifetime distribution indicating altered dynamics, and therefore excited states, between apo and iron-bound forms. Furthermore, the quenching of the steady-state and lifetimes are not equal in transitioning from apo to iron-bound, indicating non-excited state quenching is responsible for a portion iron induced quenching.
Iron release (monitoring the change in fluorescence) from recombinant isolated hTF N-lobe (residues 1-337) is reported by three Trp residues (at positions 8, 128, and 264) in two events (8.9 mid⁻¹ and 1.3 min⁻¹). Direct monitoring of iron loss via decrease in the visible absorption band at 470 nm definitively established that the faster component using intrinsic fluorescence is produced by iron release. Double Trp mutants were created to determine which of the Trp residues contributed to the iron release signal and to define the second event. Iron release from each construct at pH 5.6 revealed that both Trp128 and Trp264 are efficiently quenched when iron is bound. Based on the data from the Trp mutants, the second event was modeled as global conformational relaxation after iron removal reported by Trp128 and Trp264.
Kinetic studies on a monoferric-C-lobe construct revealed that the iron release from this lobe is slow and follows a monoexponential function with a lag ~ 50 sec. Mutation of the Trp residues in the C-lobe (at positions 344, 441, 460 and 550) indicated that the main contributor to the iron release signal is Trp460 and suggested that the lag may be ascribed to lobe-lobe cooperativity. When in the complex with the transferrin receptor, kinetic assays with the C-lobe Trp mutants revealed alterations of the Trp441 and Trp550 fluorescence emission, indicating receptor mediated conformational changes around these residues.
Using multifrequency phase fluorometry the excited-state properties of the individual Trp residues in the hTF N-lobe are reported here for the first time. These experiments were designed to investigate how iron binding affects the lifetimes of the individual Trp residues in the N-lobe and to quantitatively assign FRET efficiencies to each. The R₀ values calculated from the spectral data clearly indicated that FRET is possible. However, Trp264 undergoes dramatic changes in its lifetime distribution indicating altered dynamics, and therefore excited states, between apo and iron-bound forms. Furthermore, the quenching of the steady-state and lifetimes are not equal in transitioning from apo to iron-bound, indicating non-excited state quenching is responsible for a portion iron induced quenching.