Loop-Directed Mutagenesis of the Blue Copper Protein Amicyanin from<i>Paracoccus versutus</i>and Its Effect on the Structure and the Activity of the Type-1 Copper Site

Buning, Christian; Canters, Gerard W.; Comba, Peter; Dennison, Christopher; Jeuken, Lars J. C.; Melter, Michael; Sanders-Loehr, Joann

doi:10.1021/ja992796b

Cited by 79 publications

(100 citation statements)

References 84 publications

(112 reference 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%

mentioning

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.

Self Cite

134

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“…There have been numerous reports and reviews supporting the entatic/rack-induced state hypothesis (7,13,19,20,26,59). On the other hand, theoretical and experimental studies have questioned the role of the protein matrix in defining the geometry of the copper center (21,22,24,25). X-ray studies on the apo form of several cupredoxins, showing no changes in the ligands' conformations upon removal of the copper ion, were considered as experimental evidence of the entatic/rack-induced hypothesis for ET copper proteins (27)(28)(29)(30)(31).…”

Section: Discussionmentioning

confidence: 99%

“…This scenario, known as the entatic/rack-induced state hypothesis, was originally formulated for protein-bound cofactors by Lumry and Eyring in 1954 (16) and then extended to electron transfer metalloproteins by Malmström and Williams in the late 1960s (17,18). This concept has been matter of debate along many years (7,15,(19)(20)(21)(22)(23)(24)(25)(26). In particular, Ryde et al have reported in vacuum quantum calculations suggesting the absence of any strain in the geometry adopted by the copper in the protein framework (21,22).…”

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confidence: 99%

Flexibility of the metal-binding region in apo-cupredoxins

Zaballa

Abriata

Donaire

et al. 2012

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

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Protein-mediated electron transfer is an essential event in many biochemical processes. Efficient electron transfer requires the reorganization energy of the redox event to be minimized, which is ensured by the presence of rigid donor and acceptor sites. Electron transfer copper sites are present in the ubiquitous cupredoxin fold, able to bind one or two copper ions. The low reorganization energy in these metal centers has been accounted for by assuming that the protein scaffold creates an entatic/rack-induced state, which gives rise to a rigid environment by means of a preformed metal chelating site. However, this notion is incompatible with the need for an exposed metal-binding site and protein-protein interactions enabling metallochaperone-mediated assembly of the copper site. Here we report an NMR study that reveals a high degree of structural heterogeneity in the metal-binding region of the nonmetallated Cu A -binding cupredoxin domain, arising from microsecond to second dynamics that are quenched upon metal binding. We also report similar dynamic features in apo-azurin, a paradigmatic blue copper protein, suggesting a general behavior. These findings reveal that the entatic/rack-induced state, governing the features of the metal center in the copper-loaded protein, does not require a preformed metal-binding site. Instead, metal binding is a major contributor to the rigidity of electron transfer copper centers. These results reconcile the seemingly contradictory requirements of a rigid, occluded center for electron transfer, and an accessible, dynamic site required for in vivo copper uptake.metalloproteins | nuclear magnetic resonance | protein dynamics

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“…Recent mutagenesis studies have revealed that enhanced mobility in one of the loops of azurin does not alter the overall rigidity of the molecule (76). In addition, the ␤-barrel scaffold found in amicyanin is able to accommodate different engineered loops while maintaining the particular electronic features of the blue copper site (77). In general, the presence of a high loop content around the copper site in cupredoxins may provide a compromise solution regarding mobility.…”

Section: Discussionmentioning

confidence: 99%

Backbone Dynamics of Plastocyanin in Both Oxidation States

Bertini¹,

Bryant²,

Ciurli³

et al. 2001

Journal of Biological Chemistry

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A model-free analysis based on 15 N R 1 , 15 N R 2 , and 15 N-1 H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D 2 O exchange of amide protons and from NH-H 2 O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.

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Loop-Directed Mutagenesis of the Blue Copper Protein Amicyanin fromParacoccus versutusand Its Effect on the Structure and the Activity of the Type-1 Copper Site

Cited by 79 publications

References 84 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

Flexibility of the metal-binding region in apo-cupredoxins

Backbone Dynamics of Plastocyanin in Both Oxidation States

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