Investigating the structure and function of cupredoxins

Dennison, Christopher

doi:10.1016/j.ccr.2005.04.021

Cited by 150 publications

(198 citation statements)

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“…Similar behavior has been observed in plastocyanin (25,26), pseudoazurin (27,28), and AMI (21,22,29) and is thought to have physiological relevance (26,29). Loop mutation experiments have shown that, in most cases, lengthening the ligand-containing loop lowers the His ligand pK a (9,10,12,38), whereas loop contraction increases the value (8,11,12). In AZ, the His-117 ligand does not protonate in the accessible pH range, and a pK a of Ͻ2 has been calculated (20).…”

Section: Discussionsupporting

confidence: 59%

“…These proteins, therefore, provide a suitable system for investigating the importance of loop length and structure for the active-site integrity of a metalloprotein. Loop-directed mutagenesis has been used to swap loops between different cupredoxins, giving sites with authentic T1 properties (8)(9)(10)(11)(12). In this work, we present studies that have been aimed at assessing the structural consequences of shortening the active-site loop of a cupredoxin and have determined the shortest C-terminal loop sequence required for a functional T1 copper site.…”

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confidence: 99%

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Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Yanagisawa

Martins

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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134

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The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C 112 TFPGH 117 SALM 121 to C 112 TPH 115 PFM 118 (the native loop from the cupredoxin amicyanin) and also to C 112 TPH 115 PM 117 . The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the C 112 TPH 115 PM 117 loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the C 112 TPH 115 PFM 118 loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.copper proteins ͉ electron transfer ͉ metalloproteins ͉ protein engineering N umerous approaches are being used to design metal-binding sites in proteins (1), with many of these studies informed by an understanding of the basic structural requirements for biological metal centers. Metal-binding sites in proteins are commonly formed from loops, because these regions are reasonably tolerant to sequence modifications outside of the coordinating residues (1). Cupredoxins are copper-containing electron transfer (ET) proteins that provide a significant challenge for protein-design experiments (2-4) because their scaffold is thought to constrain the metal site structure (5). In the type 1 (T1) copper sites of cupredoxins (see Fig. 1), three of the four canonical ligands Cys, His, and, usually, Met are present on a loop linking the C-terminal strands of a rigid ␤-barrel (7, 8). The fourth ligand, a His, is donated from a ␤-strand more in the core of the fold (see Fig. 1). The lengths of the metal-binding loops in known cupredoxins range from 7 to 16 residues and have a variety of primary structures. These proteins, therefore, provide a suitable system for investigating the importance of loop length and structure for the active-site integrity of a metalloprotein. Loop-directed mutagenesis has been used to swap loops between different cupredoxins, giving sites with authentic T1 properties (8)(9)(10)(11)(12). In this work, we present studies that have been aimed at assessing the structural consequences of shortening the active-site loop of a cupredoxin and have determined the short...

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Section: Discussionsupporting

confidence: 59%

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confidence: 99%

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Yanagisawa

Martins

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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134

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“…In particular, azurin (Az) is one of the most promising ET proteins because of its very peculiar properties and its capability to form molecular complexes with natural partners (i.e., cytochrome c551) [14,15] and other biomolecules (i.e, p53) [16,17], as recently outlined. It belongs to the class of redox blue copper proteins acting as electron shuttles in many important biological reactions, for instance in photosynthesis and in bacterial respiratory redox-chains [18][19][20]. They are endowed with very efficient ET mechanisms which occur at the level of single electron, even over long distances, in a very fast, directional way [21] and can be gated by proper modulation of the applied potential [22].…”

Section: Introductionmentioning

confidence: 99%

Optical investigation of the electron transfer protein azurin–gold nanoparticle system

Delfino

Cannistraro

2009

Biophysical Chemistry

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To cite this version:Ines Delfino, Salvatore Cannistraro.Optical investigation of the electron transfer protein azurin -gold nanoparticle system. Biophysical Chemistry, Elsevier, 2008, 139 (1), pp. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. delfino@unitus.it A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT ABSTRACTThe hybrid system obtained by conjugating the protein azurin, which is a very stable and well-described The fluorescence quenching of azurin bound molecules is explained by an energy transfer from protein to metal surface and it is discussed in terms of the involvement of the Az electron transfer route in the interaction of the protein with the nanoparticle.

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“…In several cases the ligands are arranged such that an optimal geometry is precluded (1)(2)(3)(4). The resulting entatic state in a given metalloprotein is determined by the entire rigid protein scaffold in concert with the hydrogen-bonding network proximal to the coordination sphere (5,6). The particular geometry of the rack-induced state influences the electronic structure of the metal site.…”

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Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin

Zong

Wilson

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Understanding how the folding of proteins establishes their functional characteristics at the molecular level challenges both theorists and experimentalists. The simplest test beds for confronting this issue are provided by electron transfer proteins. The environment provided by the folded protein to the cofactor tunes the metal's electron transport capabilities as envisioned in the entatic hypothesis. To see how the entatic state is achieved one must study how the folding landscape affects and in turn is affected by the metal. Here, we develop a coarse-grained functional to explicitly model how the coordination of the metal (which results in a so-called entatic or rack-induced state) modifies the folding of the metallated Pseudomonas aeruginosa azurin. Our free-energy functional-based approach directly yields the proper nonlinear extrathermodynamic free energy relationships for the kinetics of folding the wild type and several point-mutated variants of the metallated protein. The results agree quite well with corresponding laboratory experiments. Moreover, our modified free-energy functional provides a sufficient level of detail to explicitly model how the geometric entatic state of the metal modifies the dynamic folding nucleus of azurin.curved chevron ͉ cupredoxin ͉ metalloproteins T he entatic state occurs in proteins when a group, metal or nonmetal, is forced into an unusual, energetically strained geometric or electronic state (rack-induced state) (1-4). Through the polypeptide's folding-induced rigidity, the protein fails to provide the expected geometry of ligating groups that would occur with freely mobile ligands in solution, thereby tuning the ligand's redox characteristics. In metalloproteins, the metal ions are typically bound to the protein through one or more lone pair donors, endogenous biological ligands (e.g., the imidazole moiety of histidine, the carbonyl oxygen of the main chain or the side chain of an asparagine residue). In several cases the ligands are arranged such that an optimal geometry is precluded (1-4). The resulting entatic state in a given metalloprotein is determined by the entire rigid protein scaffold in concert with the hydrogen-bonding network proximal to the coordination sphere (5, 6). The particular geometry of the rack-induced state influences the electronic structure of the metal site. Moreover, the resulting forced electronic structure, at least in certain cases, becomes essential for the protein's biochemical function in electron transport (7). We should remember the entatic hypothesis is in some respects still controversial. Results from some quantum calculations have suggested that the geometry of metal-ligand complexes identified as being rack-induced are not necessarily highly strained (8), whereas other theoretical studies suggest that the rigidity of the protein may in fact be much more significant than initially thought (9).Cupredoxins, a family of electron-transfer metalloproteins, are believed to adopt such a rack-induced state by way of a distorted tetrahedra...

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Investigating the structure and function of cupredoxins

Cited by 150 publications

References 323 publications

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Optical investigation of the electron transfer protein azurin–gold nanoparticle system

Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin

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