Glycine-15 in the Bend between Two α-Helices Can Explain the Thermostability of DNA Binding Protein HU fromBacillus stearothermophilus

Abstract

On the basis of sequence comparison of thermophilic and mesophilic DNA binding protein HUs, Bacillus stearothermophilus DNA binding protein HU (BstHU) seems to gain thermostability with a change in amino acid residues present on the molecular surface. To evaluate the contribution of exchange of each amino acid to the thermostability of BstHU, we constructed three mutants, BstHU-T13A (Thr13 to Ala), BstHU-G15E (Gly15 to Glu), and BstHU-T33L (Thr33 to Leu), in which the amino acids in BstHU were changed to the corresponding ones in Bacillus subtilis DNA binding protein HU (BsuHU). Stability of the mutant proteins was determined from thermal-denaturation curves. Replacement of Gly15 located in the turn region between alpha 1 and alpha 2 helices (HTH motif), with Glu (BstHU-G15E), resulted in a decrease in thermostability, and the Tm value was 54.0 degrees C compared to the Tm value of 63.9 degrees C for BstHU. The mutants, BstHU-T13A and BstHU-T33L, were, by contrast, slightly more stable (Tm values of 67.0 and 65.6 degrees C for BstHU-T13A and BstHU-T33L, respectively) than the wild type. We then generated the BsuHU mutant protein BsuHU-E15G, where Glu15 in BsuHU was in turn replaced by Gly, and we analyzed the thermostability. This substitution clearly enhanced the melting temperature by 11.8 degrees C (Tm value: 60.4 degrees C for BsuHU-E15G) compared to the value for BsuHU (Tm: 48.6 degrees C). Thus, Gly15 in the HTH motif of BstHU has an important role in the thermostability of BstHU. Characterization of the structure of the BstHU-G15E by 1H-NMR analysis showed that solvent accessibility of amide proton of Ala21 in the mutant was significantly increased compared with that of wild type, which means that the structure of the HTH motif in the N-terminal region in the mutant was changed to a more open conformation, thereby avoiding the interaction of Ala21 with either Ser17 by hydrogen bond or Ala11 by hydrophobic interaction.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

“…Table 3 shows that in most cases the difference in ⌬G stab values of hyperthermophilic and mesophilic proteins is small, usually in the range of 5 to 20 kcal/mol. Stability studies of enzyme mutants (173,261), showing that differences in ⌬G stab as small as 3 to 6.5 kcal/mol can account for thermostability increases of up to 12°C, are in complete agreement with the stability data listed in Table 3. As a consequence of the enthalpic and/or entropic stabilizations occurring in a hyperthermophilic protein, the ⌬G stabversus-T curve of this protein will be different from that of its mesophilic counterpart.…”

“…Two residues in the left-handed helical conformation, Glu15 in B. subtilis DNA binding protein HU and Lys95 in E. coli RNase H1, both in turn regions, are replaced by Gly residues in their thermophilic counterparts. Mutations Glu15Gly and Lys95Gly in B. subtilis DNA binding protein HU and E. coli RNase H1, respectively, eliminated the conformational strain created by the residues in the left-handed helical conformation, and significantly increased the thermodynamic stabilities of the two proteins (173,179). In these two examples, the stability gained by the conformational strain release was enhanced by its stabilizing effect on secondary structure interactions.…”

“…A number of attempts at stabilizing (or destabilizing) proteins by SDM have failed because they did not target protein areas that were critical for the unfolding process (173,174,337). On the other hand, mutations targeted at areas whose unfolding is limiting in the protein denaturation process can provide extensive stabilization.…”

Thermodynamic analysis of the unfolding and stability of the dimeric DNA‐binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant

“…In fact, Glu34 has been proposed to be one of the three key residues, together with Gly15 and Val42, responsible for the high thermostability of HUBst [22][23][24] and particularly of HUTmar compared to their mesophilic counterparts [4,18]. An inspection of the three-dimensional structure of the wild-type protein (Fig.…”

“…Not only has its three-dimensional structure been resolved by X-ray crystallography and NMR [19][20][21] (Fig. 1), but several mutational analyses have been made [4,[22][23][24] to find out more about the contribution of certain key amino acids to its thermostability. A new HU protein from the extreme thermophile Thermotoga maritima (HUTmar) has recently been purified and characterized [4,7,17,18].…”