Conformational Changes in Azurin from Pseudomona aeruginosa Induced through Chemical and Physical Protocols

Fuentes, Lymari; Oyola, Jessica; Fernández, Mónica; Quiñones, Edwin

doi:10.1529/biophysj.104.042580

Cited by 40 publications

(36 citation statements)

References 28 publications

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“…Az has strong charge-transfer absorption with maximum absorbance at around 625 nm due to the bond between Cu and Cys-112 [19]. The absorption band probes the oxidation state of the copper ion and other alterations around it [20]. As shown in Figure 1, Niand Co-Az have intense peak near 391 and 308 nm respectively, this is in agreement with Czernuszewicz, et al [21].…”

Section: Spectroscopic Characterization Of Cu (Ii)- Ni (Ii)-and Co (supporting

confidence: 86%

Can metal replacement increase the antitumor activity of azurin? (in vitro study)

Ali¹,

Mohamed²,

Sedek³

2017

Gen Med Open

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Azurin is a member of blue-copper proteins family which induces apoptosis in cancer cells. In the present work, azurin (Az) has been modified via replacement of copper at active site with cobalt and nickel. FTIR revealed that its secondary structure is practically indistinguishable from that of its derivatives. UV spectroscopy and DSC results revealed that native form of Az is more stable than Co-and Ni forms. SRB was used to elucidate their possible anti-tumor effect on cancer cell lines from breast and colon cells (MCF-7 and HCT116, respectively). To our best knowledge, this is the first time metal replacement of Az has been used with cell line. It appears that this process is the predominant reason for the effectiveness of Az. Experimental results showed that the anti-tumor activity of Cu-and Co Az (at concentration ≤ 25 µg/L) is higher for colon-relative to breast cells, while that for Ni-Az is higher for both cells (at low concentration, 5 µg/L). Hence, according to our findings it was revealed that Ni-Az form has the most extensive anti-antitumor effect on breast-and colon cancer cells. This highlights that Ni may be bona fide copper ion impersonator in vivo.

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Section: Spectroscopic Characterization Of Cu (Ii)- Ni (Ii)-and Co (supporting

confidence: 86%

Can metal replacement increase the antitumor activity of azurin? (in vitro study)

Ali¹,

Mohamed²,

Sedek³

2017

Gen Med Open

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“…Like WT apo-azurin (4,6,8,10,11), the variants unfold in single, reversible transitions (Fig. 1A); curves derived from CD and fluorescence data overlap for each protein, which is indicative of two-state equilibrium-unfolding processes.…”

Section: Resultsmentioning

confidence: 95%

“…The copper in azurin can be eliminated, creating apo-azurin, without change of the overall structure (3). Apo-azurin is an excellent model system because equilibrium-and kinetic-folding processes for apo-azurin are two-state reactions (4)(5)(6)(7)(8). Moreover, the stability of the apoprotein is rather high, and several mutants with native-like structure have been created (2,6,9,10).…”

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confidence: 99%

Role of structural determinants in folding of the sandwich-like proteinPseudomonas aeruginosaazurin

Wilson

Wittung-Stafshede

2005

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

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An invariant substructure that forms two interlocked pairs of neighboring ␤-strands occurs in essentially all known sandwichlike proteins. Eight conserved positions in these strands were recently shown to act as structural determinants. To test whether the residues at these invariant positions are conserved for mechanistic (i.e., part of folding nucleus) or energetic (i.e., governing native-state stability) reasons, we characterized the folding behavior of eight point-mutated variants of the sandwich-like protein Pseudomonas aeruginosa apo-azurin. We find a simple relationship among the conserved positions: half of the residues form native-like interactions in the folding transition state, whereas the others do not participate in the folding nucleus but govern high native-state stability. Thus, evolutionary preservation of these specific positions gives both mechanistic and energetic advantages to members of the sandwich-like protein family.protein folding ͉ folding transition state S andwich-like proteins are a group of very diverse proteins comprising 69 superfamilies in 38 protein folds (1). The general architecture of these proteins involves ␤-strands that form two main ␤-sheets that pack face-to-face. A recent structural and sequence analysis of the arrangements of strands within the sandwich sheets revealed a rigorously defined constraint on the supersecondary structure that holds true for 94% of all known sandwich-like structures (1). This invariant substructure consists of two interlocked pairs of neighboring ␤-strands. Within these four strands, eight hydrophobic positions (two in each strand) were found to have fixed structural roles at the interface between the ␤-sheets forming the common geometrical core. The hydrophobic residues at these eight positions are conserved across all sandwich-like proteins (1). The identification of a distinct set of structural determinants in a group of such diverse proteins as the sandwich-like proteins may shed light on how this architecture is controlled by the primary structure. Explicitly, are the eight residues invariant across all sandwichlike proteins preferentially conserved to direct the folding reaction (i.e., participating in the folding nucleus) or are they selected to stabilize the final structure? Here, we answer this question by using Pseudomonas aeruginosa azurin as our model system. P. aeruginosa azurin is a small (128 residues) single-domain protein with a ␤-barrel structure composed of eight ␤-strands, which belongs to the sandwich-like protein family (2). In vivo, a redox-active copper is coordinated to the protein, allowing for electron-transfer activity (2). The copper in azurin can be eliminated, creating apo-azurin, without change of the overall structure (3). Apo-azurin is an excellent model system because equilibrium-and kinetic-folding processes for apo-azurin are two-state reactions (4-8). Moreover, the stability of the apoprotein is rather high, and several mutants with native-like structure have been created (2, 6, 9, 10). Apo-azurin folds ...

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“…With a few exceptions [55,56], encapsulation of proteins in nanoporous silica gels normally produces an increased stability with respect to thermal and chemical insults, or long-term storage [9]. At the same time, encapsulation allows to selectively stabilize specific subsets of protein conformations, that, depending on encapsulation conditions and the protein system, can either reflect the "natural" distribution of species under defined solution conditions [57,58], or an artificially biased population (e.g., the highly reactive conformation of an enzyme encapsulated as a biocatalyst or bioreactor [59]).…”

Section: Effect Of Silica Gel Encapsulation On Thermodynamic Stabilitmentioning

confidence: 99%

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

Ronda

Bruno

Campanini

et al. 2015

COC

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Abstract:The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulation within the pores of a silica gel three-dimensional network, the mechanism of interaction between the protein and the gel matrix, and its effects on protein structure, function, stability and dynamics. The main applications in the field of biosensor design are described. Special emphasis is devoted to silica gel encapsulation as a tool to selectively stabilize subsets of protein conformations for biochemical and biophysical studies, an application where silica-based encapsulation demonstrated superior performance with respect to other immobilization techniques.

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Conformational Changes in Azurin from Pseudomona aeruginosa Induced through Chemical and Physical Protocols

Cited by 40 publications

References 28 publications

Can metal replacement increase the antitumor activity of azurin? (in vitro study)

Can metal replacement increase the antitumor activity of azurin? (in vitro study)

Role of structural determinants in folding of the sandwich-like proteinPseudomonas aeruginosaazurin

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

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