Trp128Tyr mutation in the N-lobe of recombinant human serum transferrin: 1H- and 15N-NMR and metal binding studies.

The conserved Trp residue within helix 5 of the N-lobe of human serum transferrin (hTF/2N, 40 kDa) has been mutated to Tyr. NMR and CD spectra and energy calculations show that the mutation causes little perturbation of the overall structure of hTF/2N although the chelating agent Tiron removed Fe3+ from the mutant protein about three times faster than from wild-type hTF/2N. 1H-NMR resonances of residues in the Leu122-Trp128-Ile132 hydrophobic patch are assigned both by ring current calculations and with the aid of the mutation. [1H, 15N]-NMR resonances for 11 of the 14 Tyr residues were observed in the spectra of 15N-Tyr-hTF/2N and a resonance for Tyr128 was assignable in spectra of the mutant. The 15N resonance of Y128 was sensitive to oxalate and Ga3+ binding, and Ga3+ binding perturbed 15N resonances for most of the Tyr residues. Since these are well distributed over the N-lobe, it can be concluded that metal-induced structural changes are not merely local to the binding site.

Interlobe communication in 13C-methionine-labeled human transferrin.

[1H, 13C] NMR investigations of metal-induced conformational changes in the blood serum protein transferrin (80 kDa) are reported. These are thought to play an important role in the recognition of this protein by its cellular receptors. [1H, 13C] NMR resonance assignments are presented for all nine methionine 13CH3 groups of recombinant deglycosylated human transferrin on the basis of studies of recombinant N-lobe (40 kDa, five Met residues), NOESY-relayed [1H, 13C] HMQC spectra, and structural considerations. The first specific assignments for C-lobe resonances of transferrin are presented. Using methionine 13CH3 resonances as probes, it is shown that, with oxalate as the synergistic anion, Ga3+ binds preferentially to the C-lobe and subsequently to the N-lobe. The NMR shifts of Met464, which is in the Trp460-centered hydrophobic patch of helix 5 in the C-lobe in contact with the anion and metal binding site, show that Ga3+ binding causes movement of side chains within this helix, as is also the case in the N-lobe. The C-lobe residue Met382, which contacts the N-lobe hinge region, is perturbed when Ga3+ binds to the N-lobe, indicative of interlobe communication, a feature which may control the recognition of fully-metallated transferrin by its receptor. These results demonstrate that selective 13C labeling is a powerful method for probing the structure and dynamics of high-molecular-mass proteins.

A modified technique for evaluating apical base relationships.

1. The ANB difference is not always an accurate method of establishing the actual amount of apical base divergence. In addition to the geometric shortcomings previously pointed out, the results of this study indicate that the ANB landmarks are subject to growth and treatment mechanics which cause corresponding variations in the measurements used. 2. The AXD method described in this study demonstrates a method of relating apical bases which eliminates two of the variables, nasion and point B. The AXD angle is a more critical evaluation and was found to correspond significantly with a linear measurement of the actual apical base difference. 3. This study and others have demonstrated conclusively that angular measurements cannot compensate for divergence of apical bases resulting from variations in vertical facial height. For this reason, a set of linear measurements was proposed which would offer an accurate method of evaluating the pre- and posttreatment changes taking place. 4. The linear measurements introduced in this study demonstrate an accurate method of assessing apical base relationships. The use of these measurements provides a critical evaluation of treatment and places proper emphasis on favorable growth and treatment mechanics. 5. The study of the pre- and posttreatment linear measurement concerning point A determined that this landmark remained remarkably constant during orthodontic treatment. It would appear from this study that treatment mechanics for Class II cases do not actively relocate point A but actually hold it in position while the middle face is growing forward. 6. The A-D' measurement is concerned with the actual distance, in millimeters, between the apical base landmarks. In the fifty cases studied, the mean pretreatment measurement of A-D' was reduced 15.8 per cent as the Class II malocclusion was corrected. The mean pretreatment ANB underwent a reduction of 43.1 per cent on these same cases. While the A-D' pre- and posttreatment change may not be as dramatic as the ANB evaluation, the major point to consider is the critical degree of accuracy which this measurement brings into an analysis.