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Biophysical studies on a dimeric hemoglobin and on TATA binding protein·DNA complexes

Jiong Wu, University of Nebraska - Lincoln

Abstract

The kinetics and thermodynamics of CO binding to the dimeric hemoglobin I of Noetia ponderosa were studied. CO recombination following laser photolysis was analyzed to resolve the recombination pathway and obtain the associated allosteric rate constants. Spectroscopy was used to obtain the Hill number and P50. Isothermal titration calorimetry was used to determine ΔH°, ΔG° and ΔS° for CO binding at 20°C. These data were analyzed in terms of the allosteric model. ^ Time-resolved fluorescence emission measurements, in conjunction with fluorescence resonance energy transfer (FRET), were used to determine the distance distributions, P(R), for free and TBP-bound DNA oligomers bearing TATA sequences. TBP is the TATA binding protein. The derived bend angle for the reference sequence, AdMLP, is 76.2° for a two-kink bend model, in excellent accord with the corresponding co-crystal structure. Bend angles for the five mutant sequences vary from 29.9° for A3 to 61.8° for T5, in contrast to the conserved 80° bends in the corresponding co-crystals. These solution DNA bend angles are strongly correlated with the transcription activities. The derived bend angles for variant sequences, C7 and T6, are significantly increased in the presence of osmolyte, however, there is little effect of osmolytes on the solution geometry of AdMLP or the transcriptionally inactive variant, A3. A two-state model was used to accommodate the sequence- and osmolyte concentration dependence of DNA bending. Water release in osmolyte solutions was subsequently studied in terms of osmotic stress theory. That theory did not satisfactorily explain the results. ^ The intrinsic curvatures and flexibilities of free DNA duplexes were also studied by FRET. The TATA element and A6 tract impart fixed curvatures. The repeated An (n ≥ 2) runs correspond to increased rigidity for free duplexes. ^

Subject Area

Chemistry, Biochemistry|Biophysics, General

Recommended Citation

Wu, Jiong, "Biophysical studies on a dimeric hemoglobin and on TATA binding protein·DNA complexes" (2001). ETD collection for University of Nebraska - Lincoln. AAI3022672.
http://digitalcommons.unl.edu/dissertations/AAI3022672

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