Thermodynamic analysis of the single-stranded DNA binding activity of the archaeal replication protein A (RPA) from Sulfolobus solfataricus.
The single-stranded DNA binding protein from Sulfolobus solfataricus (Sso-RPA) binds single-stranded DNA with dissociation constants in the range of 10-30 nM at room temperature. The affinity for DNA decreases at higher temperatures. At 85 degrees C, the optimal growth temperature of the crenarchaeot S. solfataricus, the dissociation constant is only about 1 microM. We analyzed the equilibrium between Sso-RPA and a fluorescently labeled 13 nucleotide oligonucleotide by fluorescence anisotropy measurements in the presence of four different salts and in the temperature range between 10 and 60 degrees C. In the presence of potassium chloride and choline chloride, three to four ions are released upon complexation, independent of the temperature. In contrast, in the presence of potassium fluoride and potassium glutamate, we observed a significant change of the number of ions released when the temperature was varied. The binding reaction is strongly exothermic with enthalpies of about -55 to -70 kJ/mol, depending upon the salt. Van't Hoff analysis suggests that the binding enthalpy is temperature independent.
Kernchen
Uwe
U
Institute of Biochemistry, University of Bayreuth, Universitätstrasse 30, 95447 Bayreuth, Germany.
Lipps
Georg
G
D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D019641:DNA, Archaeal / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D007700:Kinetics; D011189:Potassium Chloride / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D048229:Sulfolobus solfataricus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005971:Glutamates; D011994:Recombinant Proteins; D051716:Replication Protein A; D012492:Salts; D011189:Potassium Chloride; D002794:Choline