PubMed Health⌕ Search

PubMed · 8513891

Engineering type 1 copper sites in proteins.

Abstract

The use of site-directed mutagenesis methods has revolutionalized the study of the so-called type 1 and type 2 copper sites in proteins. In particular our understanding of the relation between the structure, and the mechanistic and spectroscopic features of these sites is benefitting from the application of these techniques. Recent progress in the field is reviewed with emphasis on the study of type 1 sites. Topics covered comprise the characteristics of the natural type 1 and type 2 sites, the genetics of blue copper proteins, the modification of Cu sites, the spectroscopy of natural and engineered type 1 and type 2 sites, the effect of mutations on midpoint potentials and the mechanism of electron transfer as carried out by the blue copper proteins.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

G W Canters, G Gilardi. 1993-06-28. Engineering type 1 copper sites in proteins.. https://doi.org/10.1016/0014-5793(93)81410-2

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

Engineering copper sites in proteins: loops confer native structures and properties to chimeric cupredoxins.

The ligand-containing loops of two copper-binding electron-transfer proteins (cupredoxins) have been swapped. In the azurin (AZ) variant in which the plastocyanin (PC) sequence is introduced (AZPC), the loop adopts a conformation identical to that in PC. The reduction potential of AZPC is raised as compared to AZ and matches that of PC. In the previously published AZAMI variant (AMI = amicyanin), the shorter introduced loop adopts the same conformation as in AMI, and the reduction potential is lowered to equal that of AMI (Yanagisawa, S.; Dennison, C. J. Am. Chem. Soc. 2004, 126, 15711-15719. Li, C.; et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7258-7263). Thus, the loop structure plays an important role in tuning the reduction potential of a type 1 copper site with contributions from protein dipoles in this region probably the most important feature. The structure of the loop also seems to be a major factor in controlling dissociation and protonation of the C-terminal His ligand, which can act as a switch to regulate electron-transfer reactivity. The PCAZ variant (PC with the AZ loop) possesses an active site, which is different from those of both PC and AZ, and it is assumed that the introduced loop does not adopt a structure as in AZ. This contributes to the observed instability of PCAZ and highlights that loop-scaffold interactions are important for stabilizing the active site of a cupredoxin.

Azurin↗

Conserved water molecular dynamics of the different X-ray structures of rusticyanin: an unique aquation potentiality of the ligand bonded Cu++ center.

The invariant water molecular interaction involving in the Rusticyanin of Thiobacillus ferrooxidans is thought to be important for its molecular complexation with other proteins at differential acidophilic situation. The comparative analysis of the different x-ray, energy minimized, and auto solvated structures of Rusticyanin revealed the presence of five specific invariant bound water molecules (among the approximately 150 water molecules per monomer) in the crystals. The five W 205, W 206, W 112, W 214, and W 221 water molecules (in Rusticyanin PDB code: 1RCY) were seem to be invariant in all the seven structures (PDB codes: 1RCY, 1A3Z, 1A8Z, 1E3O, 1GY1, 1GY2, 2CAL). Among the five conserved water molecules the W 221 (of 1 RCY or the equivalent water molecules in the other oxidized form of Rusticyanin structures) had endowed an interesting coordination potentiality to Cu(+2) ion during the energy minimization. The W 221 was observed to approach toward the tetrahedrally bonded Cu(+2) ion through the opposite (or trans) route of metal-bonded Met 148. This direct water molecular coordination affected the tetrahedral geometry of Cu(+2) to trigonal bipyramidal. Presumably this structural dynamics at the Cu(+2) center could involve in the electron transport process during protein-protein complexation.

Azurin↗

Protein stability in ice.

This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins.

Azurin↗