Effect of circular permutation on the structure and function of type 1 blue copper center in azurin

Abstract

Type 1 copper (T1Cu) proteins are electron transfer (ET) proteins involved in many important biological processes. While the effects of changing primary and secondary coordination spheres in the T1Cu ET function have been extensively studied, few report has explored the effect of the overall protein structural perturbation on active site configuration or reduction potential of the protein, even though the protein scaffold has been proposed to play a critical role in enforcing the entatic or "rack-induced" state for ET functions. We herein report circular permutation of azurin by linking the N-and C-termini and creating new termini in the loops between 1 st and 2 nd b strands or between 3 rd and 4 th b strands. Characterization by electronic absorption, electron paramagnetic spectroscopies, as well as crystallography and cyclic voltammetry revealed that, while the overall structure and the primary coordination sphere of the circular permutated azurins remain the same as those of native azurin, their reduction potentials increased by 18 and 124 mV over that of WTAz. Such increases in reduction potentials can be attributed to subtle differences in the hydrogenbonding network in secondary coordination sphere around the T1Cu center.

Engineering the metal-binding loop at a type 1 copper center by circular permutation

“…In a previous study, two construct have been built for circular permutation in Az. 27 The mutants, cpAz1 and cpAz2, maintain typical T1C spectroscopic properties, suggesting the robustness of Az structure. However, as the metalbinding loop harbors 3 ligands of copper center, the question remains whether circular permutation will perturb the copper center.…”

“…2B, here we applied circular permutation strategy, to connect N-and C-termini with a linker GIPGG and create new termini at Gly116 (N-terminus) and Phe114 (C terminus), similar to what have been reported for cpAz1 and cpAz2. 27 The Az-loop mutant was constructed by overlap-extension PCR. The protein was puried in a procedure similar to that of the WT protein 28 to obtain metal-free apo protein with a yield of $40 mg L À1 .…”

“…Colors of the secondary structures are matched between crystal structure and the scheme. Adopted from ref 27. with permission from Wiley.…”

CuA-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential

“…3 The rst two types of centers share the cupredoxin fold and the interesting feature that all but one of the coordinating amino acids are located in a single loop that connects two b-strands. 1,4 Therefore, along with point mutations, replacement of the entire ligand loop by sequences from other proteins or unnatural sequences, 5 has become one of the preferred strategies for modulating the electronic properties of T1 4,[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] and Cu A sites. [24][25][26][27][28][29][30] This methodology has also allowed for the successful insertion of Cu A sites into the scaffold of T1 [31][32][33][34][35] proteins and vice versa.…”

Circular permutation of a bacterial tyrosinase enables efficient polyphenol‐specific oxidation and quantitative preparation of orobol

“…Most recently, Yu et al (2017) also reported the successful CP of azurin, one of type 1 copper proteins with important roles in electron transfer in diverse biological systems. However, there has been no reports of CP of tyrosinases such as BmTy which have a binuclear type 3 copper center located at the core of a four-helical bundle (Sendovski, Kanteev, Ben-Yosef, Adir, & Fishman, 2011).…”