Structural Elements Model Applied To Analyze The Stability Of Azurin

Azurin（Az）is “type I” copper metallo proteins that are utilized in plants and bacteria for electron transfer.It is a single polypeptide chain composed of 128 amino acid residues,the secondary structure of the protein consists of α-helix and eight β strands packed in sandwich structure and where the single tryptophan(Trp⁴⁸)buried in.Central copper ion in the protein is coordinated by three equatorial residues（His 117,His 46 and Cys 112）and two more weakly bound axial ligands（Gly 45 and Met 121）.For the unusual geometric structure of the copper binding site,the highly coordination covalent nature of the copper-Cys112 bond gives Cu^Ⅱ-Az a characteristic ligand to metal charge transfer（LMCT）band centered around 625 nm,and the fluorescence quenching dramatically accompanies Cu^Ⅱ binding to the protein.Based on the reasons mentioned above,the reaction between apo-Az and Cu^Ⅱ-Az were studied by spectroscopy methods.The fluorescence of apo-Az was quenched quickly after the adding of Cu^Ⅱ,and we speculate it is the process ofCu^Ⅱ captured by a amino acid on the surface of protein.But the ultraviolet-visible spectra showed that the absorbance at 625 nm increased tardily,which indicate the formation of Cu^Ⅱ-Az.The unfolding process of apo-Az,AgI-Az,ZnⅡ-Az,CuI-Az and Cu^Ⅱ-Az induced by Gu HCl were also studied by spectroscopy and the experimental datas were analyzed according to structure element model.The result showed that,Az is consists of two structural elements,the only tryptophan residues(Trp⁴⁸)is locate in the structural element 1 and the active cite（metal binding site）is locate in the structural element 2.The unfolded curve of apo-Az obeyed a two-state transition because the two structural elements were equivalent.The two structural elements in apo-Az cannot be distinguished by AgI,it is due to thatAgI have the same effect on the stability of both structural elements and the unfolded curve of AgI-Az also obeyed two-state.ZnⅡ,CuI and Cu^Ⅱ can increase the stability of the two structural elements in different degree and the unfolded curves of M-Az（M = ZnⅡ,CuI and Cu^Ⅱ）best fit to three-state transition.The intermediate state can be considered as the structural element 1 has been destroyed,whereas the structural element 2 retains the native structure.Further,SDS and CTAB induced unfolding process of apo-Az and Cu^Ⅱ-Az were investigated by spectroscopy.The results showed that the unfolding curve of apo-Az is a typical two-state transition but the fluorescence spectra indicate the unfolding degree of apo-Az induced by SDS and CTAB is less than GuHCl.The unfolding curve of Cu^Ⅱ-Az induced by SDS show three-state because two structure elements both unfolded.However,CTAB gather to micelle easily because the critical micelle concentration was only 0.16 mM,which reduced the ability of unfolding protein induced by CTAB.In addition,compared to SDS,CTAB taking a positive charge,it is harder for CTAB to attack Cu^Ⅱ at the active site for the positive charge.Thus,CTAB only induced in unfolding of structure element 1 in Cu^Ⅱ-Az and the unfolding curve fit two-state.