Halophilic adaptation of protein-DNA interactions.

Pyrococcus woesei ( Pw ) is an archaeal organism adapted to living in conditions of elevated salt and temperature. Thermodynamic data reveal that the interaction between the TATA-box-binding protein (TBP) from this organism and DNA has an entirely different character to the same interaction in mesophilic counterparts. In the case of the Pw TBP, the affinity of its interaction with DNA increases with increasing salt concentration. The opposite effect is observed in all known mesophilic protein-DNA interactions. The halophilic behaviour can be attributed to sequestration of cations into the protein-DNA complex. By mutating residues in the Pw TBP DNA-binding site, potential sites of cation interaction can be removed. These mutations have a significant effect on the binding characteristics, and the halophilic nature of the Pw TBP-DNA interaction can be reversed, and made to resemble that of a mesophile, in just three mutations. The genes of functionally homologous proteins in organisms existing in different environments show that adaptation is most often accompanied by mutation of an existing protein. However, the importance of any individual residue to a phenotypic characteristic is usually difficult to assess amongst the multitude of changes that occur over evolutionary time. Since the halophilic nature of this protein can be attributed to only three mutations, this reveals that the important phenotype of halophilicity could be rapidly acquired in evolutionary time.

Site-specific cation binding mediates TATA binding protein-DNA interaction from a hyperthermophilic archaeon.

Pyrococcus woesei (Pw) is a hyperthermophilic archaeal organism that exists under conditions of high salt and elevated temperature. In a previous study [O'Brien, R., DeDecker, B., Fleming, K., Sigler, P. B., and Ladbury, J. E., (1998) J. Mol. Biol. 279, 117-125], we showed that, despite the similarity of primary and secondary structure, the TATA box binding protein (TBP) from Pw binds thermodynamically in a fundamentally different way to its mesophilic counterparts. The affinity of the interaction increases as the salt concentration is increased. The formation of the protein-DNA complex involves the release of water and the uptake of ions, which were hypothesized to be cations. Here we test this hypothesis by selecting potential cation binding sites at negatively charged, acidic residues in the complex interface. These were substituted using site-directed mutagenesis of specific residues. Changes in the thermodynamic parameters on formation of the mutant protein-DNA complex were determined using isothermal titration calorimetry and compared to the wild type interaction. Removal of a glutamate residue from the binding site resulted in the uptake of one less cation on formation of the complex. This glutamate (E12) is directly involved in the binding of cations in the complex interface. Substitution of another acidic residue proximal to the DNA binding site (D101) had no effect on cation uptake, suggesting that the location of the amino acid on the protein surface is important in dictating the potential to coordinate cations. Removal of the cation binding site provided a more favorable entropy of binding; however, this effect is significantly reduced at higher salt concentrations. The removal of the cation binding site led to an increase in affinity with respect to the wild-type TBP at low salt concentrations.

5-(1-propargylamino)-2'-deoxyuridine (UP): a novel thymidine analogue for generating DNA triplexes with increased stability.

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (UP). The intra-molecular triplex A6-L-T6-L-(UP)5T (L = two octanediol residues) shows a single UV-melting transition which is >20 degrees higher than that of the parent triplex A6-L-T6-L-T6at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by UP, in a pH dependent fashion. Triplex stability increases by approximately 12 K for each T to UP substitution. Quantitative footprinting studies have examined the interaction of three UP-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAGAGAAAAAAGAA. At pH 5.0, the UP-containing oligo-nucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (UP)6CUPT binds approximately 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.

The contribution of cytosine protonation to the stability of parallel DNA triple helices.

The influence of the position of the CG.C+ triplet and the contribution of protonation at the N3 of the Hoogsteen cytosine residue on the stability of various sequences of parallel triple helices having the general composition d[(A5G)-x-(T5C)-x-(T5C)] and d[(A4G2)-x-(T4C2)-x-(T4C2)], where x is the hexaethylene glycol linker, has been determined by NMR, ultraviolet melting and absorbance spectrophotometry. The apparent pK value, i.e. the pH at which the observable has changed by 50% of its range, was typically in the range 6 to 7. However, the NMR spectra unequivocally showed that the pK of the protonated cytosine residue must be at least 9.5 for internal positions. This is five units above the pK of the free nucleotide, and represents a free energy of stabilisation from protonation of >11.5 RT. The pK of terminal cytosine residues is much lower, in the range 6.2 to 7.2, accounting for a free energy of stabilisation from protonation of 3.6 to 6 RT. The van't Hoff enthalpies were determined for the dissociation of the protonated triplex into the duplex+strand, and for the duplex to strand transition. The mean value for the duplexes were 23 to 27 kJ mol-1 base-pair, and 25 to 30 kJ mol-1 for the triplexes containing internal CG.C+ triplets. Good agreement was obtained for the thermodynamic parameters by the different methods. Free energy differences for the transition between the protonated triplex and the duplex+protonated strand were calculated at 298 K. The DeltaG of stabilisation of an internal CG.C triplet compared with a terminal CG.C triplet was about 6 kJ mol-1 ; a similar stabilisation was observed for the triplexes containing two CG.C triplets compared with those containing a single CG.C triplet. The very large stabilisation from protonation is too large to be accounted for by a single hydrogen bond, and is likely to include contributions from electrostatic interactions of the positive charge with the phosphate backbone, and more favourable interactions between neighbouring bases owing to the very different electronic properties of the protonated C.