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

Abstract

We have studied the stability of the histone-like, DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant by differential scanning microcalorimetry and CD under acidic conditions at various concentrations within the range of 2-225 lM of monomer. The thermal unfolding of both proteins is highly reversible and clearly follows a two-state dissociation/unfolding model from the folded, dimeric state to the unfolded, monomeric one. The unfolding enthalpy is very low even when taking into account that the two disordered DNA-binding arms probably do not contribute to the cooperative unfolding, whereas the quite small value for the unfolding heat capacity change (3.7 kJAEK ) stabilizes the protein within a broad temperature range, as shown by the stability curves (Gibbs energy functions vs. temperature), even though the Gibbs energy of unfolding is not very high either. The protein is stable at pH 4.00 and 3.75, but becomes considerably less so at pH 3.50 and below, to the point that a simple decrease in concentration will lead to unfolding of both the wild-type and the mutant protein at pH 3.50 and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more stable than its E34D mutant, particularly at pH 4.00 and 3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJAEmol )1 at 25°C for DDG at pH 4.00, 3.75 and 3.50, respectively), which seems to be related to the effect of a salt bridge between E34 and K13.Keywords: differential scanning microcalorimetry; hyperthermophilic HU protein; polar interactions; thermal stability; unfolding heat capacity.Proteins from thermophilic and hyperthermophilic microorganisms are of major interest to industrial biotechnology because they are usually more stable at high temperatures than their analogues from mesophilic organisms whilst they retain the folding patterns of their protein family [1][2][3][4]. Most attempts at discovering the origin of their stability have involved comparative thermodynamic and/or amino acid sequence analyses of homologous proteins from organisms living at different temperatures [1,2,[5][6][7][8][9]. Thus it is generally accepted that to arrive at a complete understanding of the thermal adaptation strategies of these proteins it is necessary to obtain and compare the unfolding thermodynamic functions of mutants and other family members.HU is a small histone-like bacterial protein that binds to DNA. It is abundant in all prokaryotes and its sequence is quite similar in a considerable number of species [10]. It is essential in the assembly of supramolecular nucleoprotein complexes and is also involved in a variety of DNA metabolic events, such as replication, transcription and transposition [11,12]. Its ability to repair DNA [13,14] and to prevent DNA duplex melting [7] has also been described.HU proteins from several species of bacillus growing in environments of different temperatures have already been isolated and st...

A Miniprotein Scaffold Used to Assemble the Polyproline II Binding Epitope Recognized by SH3 Domains

“…These experimental results were fitted simultaneously with the DSC data using the multiple curve-fitting procedure (see Materials and Methods). 42 The quality of the multidimensional analysis ( Figure 3) confirms the validity of the proposed two-state unfolding/dissociation model and reveals some interesting results. Our main observation, taking 120 mM as a reference concentration, is that the melting temperature obtained for APP-RP1 is about 17 deg.…”

“…The simplest applicable model should be that of a two-state unfolding/dissociation process in which only the native dimeric, N 2 , and the unfolded monomeric, U, states are populated to any extent in solution (equation (1)). 42 Together with the DSC data, we used CD to follow the thermal unfolding. As stated above, far-UV and near-UV spectra obtained at 5 8C and 95 8C reveal clear differences between the N and U conformations.…”

Structural basis of the high thermal stability of the histone-like HU protein from the mollicute Spiroplasma melliferum KC3

“…Previous studies of the thermal denaturation of HU proteins from E. coli , B. subtilis , and T. volcanium showed that the melting point increases with increasing protein concentration and increasing ionic strength of the solution, which suggests a substantial contribution of hydrophobic interactions to the stability of HU dimers142125 (Table 2). In addition to hydrophobic interactions at the dimer interfaces, the role of other structural factors (e.g., hydrogen bonds and salt bridges) in the thermal stability of HU proteins has also been discussed in a number of reports141920212223242526. For example, it was shown that residues Gly15, Glu34, and Val42 (numbering corresponds to the sequence of the T. maritima protein) are responsible for high thermal stability of HU proteins from T. maritima and B. stearothermophilus 20222426.…”

“…In addition to hydrophobic interactions at the dimer interfaces, the role of other structural factors (e.g., hydrogen bonds and salt bridges) in the thermal stability of HU proteins has also been discussed in a number of reports141920212223242526. For example, it was shown that residues Gly15, Glu34, and Val42 (numbering corresponds to the sequence of the T. maritima protein) are responsible for high thermal stability of HU proteins from T. maritima and B. stearothermophilus 20222426. The replacement of Gly15 with Glu led to a substantial decrease in the thermal stability of the protein due to destabilization of the HTH domain structure1924.…”

“…All HU dimers feature a compact architecture, including several intertwined α-helices, two three-stranded β-sheets, and two disordered arms that are flexible in the absence of DNA19. Due to the existence of HU homologues possessing moderate14202122 or high19202223242526 thermal stability, the small size of the proteins, and the similarity of the three-dimensional structures, HU proteins may serve as a convenient model for investigating the structural basis of thermal stability. It has been shown that thermal denaturation of HU proteins usually occurs through dissociation of the dimer into denatured (unfolded) monomers182225 with the only known exception being the E. coli HU protein, which undergoes a two-step denaturation described by the dimer – dimeric intermediate – denatured monomers model14.…”