A new model used to calculate the free energy change of protein unfolding is presented.In this model,proteins are considered to be composed of structural elements.The unfolding of a structural element obeys a two-state mechanism and the free energy change of the element can be obtained by a linear extrapolation method.If a protein consists of the same structural elements,its unfolding will displays a two-state process,and only the average structural element free energy change < △G 0 element(H2O)> can be measured.If protein consists of completely different structural elements,its unfolding will show a multi-state behavior.When a protein consists of n structural elements its unfolding will shows a(n+1)-state behavior.A least-squares fitting can be used to analyze the contribution of each structural element to the protein and the free energy change of each structural element can be obtained by using linear extrapolation to zero denaturant concentration,not to the start of each transition.The measured △G0 protein(H2 O) is the sum of the free energy change for each structural element.Using this new model,we can not only analyze the stability of various proteins with similar structure and similar molecular weight,which undergo multi-state unfolding processes,but also compare the stability of proteins with different structures and molecular weights using the average structural element free energy change < △G0 element(H2O)>.Although this method cannot completely provide the exact free energy of proteins,it is better than current methods.
The binding properties of ethylene-N,N-dianthranilate(EDA)and Ni(II)were studied by difference UV-Vis in 0.05 M Tris-HCl buffer(pH 7.4).The stoichiometric ratio of EDA to nickel(1:1)was confirmed and the conditional binding constant(logKNi-EDA=12.33±0.06)was obtained.In addition,an interesting tetranuclear complex[Ni4(EDA)4]·2CH3CH2OH·9H2O was obtained unexpectedly in alcohol-water solution and characterized by X-ray crystallography and electrospray ionization mass spectra(ESI-MS).It is found that EDA acts as a pentadentate ligand and performs a chelating-bridging coordination mode.
Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin.The first amino acid of the Ca2+-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue.The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen.Aromatic-sensitized Tb3+fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation.Based on fluorescence titration curves of Lu2-D37K,the conditional binding constants of the EoCen loop II were quantitatively found to be KII=(1.61±0.04)×105 L mol-1 and KII=(3.52±0.08)×102 L mol-1 with Tb3+ and Ca2+,respectively.Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe,exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant.
LIU WenDUAN LianZHAO YaQinLIANG AiHuaYANG BinSheng
In the current three-state protein unfolding model, the two transitions are considered to be independent and each transition is fitted to a two-state unfolding model. This three-state unfolding process is therefore composed of two sequential two-state unfolding processes. In this paper, a modified method is presented to determine the value of the unfolding free energy [Gt0otal(H2O)] for the three-state unfolding equilibrium of proteins. This method is demonstrated on the apoCopC protein mutant, Y79W-W83F-Cu, which unfolds via a three-state process. The value of Gt0otal(H2O) calculated using the modified method was found to be more accurate in determining Gt0otal(H2O) than the previously reported method.
