Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 343, Issue 2, Pages 339-360Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2004.08.042
Keywords
isothermal titration calorimetry; fluorescence; DNA-binding protein
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Funding
- NIGMS NIH HHS [GM49686] Funding Source: Medline
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Sac7d is a hyperthermophile chromatin protein which binds nonspecifically to the minor groove of duplex DNA and induces a sharp and kink of 66degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25degreesC (K-a=3.5(+/-1.6)x10(6) M-1) and increased by nearly an order of magnitude from 10degreesC to 80degreesC. The enthalpy of binding was unfavorable at 25degreesC, and decreased linearly from 5degreesC to 60degreesC. A positive binding heat at 25degreesC is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6degreesC) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25degreesC with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25degreesC. In addition, an anomalous positive DeltaC(p) of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a T-m of 15-20degreesC and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion. (C) 2004 Elsevier Ltd. All rights reserved.
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