Probing The Structure-function Relationship Of Type ? Copper Protein By Circular Permutation

Copper protein is divided into three types:type ?,? and ?.Type ? copper protein is involved in many important biological electron transfer,including photosynthesis and respiration.It is a copper-containing protein family with small molecular weight.The most representative member of type ? copper protein is Azurin from Pseudomonas aeruginosa.To study the relationship of structure and function in type ? copper protein,we constructed a mutant of Azurin,cpAz3 by creating new termini between the 6st and 7nd ? strand by circular permutation,we characterized the mutant by the spectroscopic methods,crystallography and electrochemistry of the protein to study the relationship between the active center of the protein and the structure.Absorption spectrum of the protein showed a ?max of 624 nm by UV-Vis spectroscopy,only slightly blue-shifted from that of WTAz(627 nm).The mutant,cpAz3,exhibited absorption spectrum typical of blue copper proteins.It shows that the binding capacity of Cu2+ is similar to that of the wild type Azurin.EPR spectrum of cpAz3 showed more than one species.Simulation of the spectrum showed the majority of the signal(84%)is from a type ? site with the same parameters as WT Az,while the rest of the signal is from a type ?site is hard to determine the exact structure of the species.For crystallographic characterization,I analyzed the crystal structure and determined the distance between the copper center and the ligand,I found that the overall structure of the protein have not changed.At the primary coordination sphere,the His65-Cu distance is 0.07 A and Met-Cu distance is 0.11 A longer than these in the WT structure.At the second coordination sphere,only distance between N? of N66 and S? of Cys131 is 0.1 A longer.These distances are within the normal range observed in structures in WT Azurin.Changes in these distances lead to changes in the redox potential of proteins.For electrochemical characterization,the redox potential of cpAz3 was measured by cyclic voltammetry using a carbon nanotube modified glassy carbon electrode.Redox potential cpAz3 was determined to be 353±2 mV and 239±12 mV at pH 4.0 and pH 7.0,respectively,which is 29 mV and 13 mV higher than that of the WT.By studying the spectral properties,electrochemistry and crystallography of cpAz3,it exhibited characterization typical of blue copper proteins.These dates indicating circular permutation has minimal perturbation to the structure.