Expression of the blue copper protein azurin from <i>Pseudomonas aeruginosa</i> in <i>Escherichia coli</i>

Karlsson, Bengt; Pascher, Torbjörn; Nordling, Margareta; Arvidsson, Rolf H. A.; Lundberg, Lennart

doi:10.1016/0014-5793(89)80285-6

Cited by 111 publications

(90 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Samples of azurin and apo-and holoCu A proteins were uniformly labeled with 15 N or 13 C and 15 N (Cambridge Isotope Laboratories, Inc.) and produced as described elsewhere (40,47,64). The apoCu A protein was expressed and purified in the absence of copper, whereas azurin was purified as Cu(II)-azurin and the apoprotein was obtained by dialysis against 50 mM KCN, 200 mM Tris·HCl pH 8.5, followed by several dialysis steps without the complexing agent.…”

Section: Methodsmentioning

confidence: 99%

Flexibility of the metal-binding region in apo-cupredoxins

Zaballa

Abriata

Donaire

et al. 2012

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

View full text Add to dashboard Cite

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

show abstract

Section: Methodsmentioning

confidence: 99%

Flexibility of the metal-binding region in apo-cupredoxins

Zaballa

Abriata

Donaire

et al. 2012

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

View full text Add to dashboard Cite

show abstract

“…CuCI2 (1001aM), ampicillin (100mgl -~) and isopropyl-#-D-thiogalactopyranoside (IPTG, 0.5mM) were added when the cultivation was started. The purification of Met l21Ala was carried out according to Karlsson, Pascher, Nordling, Arvidsson & Lundberg (1989), except that the sample was gel-filtrated using Sephadex S-100 gel in 100mM phosphate buffer. The sample was checked on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) denaturing gel with #-mercaptoethanol, showing that the sample only contained azurin.…”

Section: Dna Techniques Expression and Purificationmentioning

confidence: 99%

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

Tsai¹,

Bonander²,

Harata³

et al. 1996

Acta Cryst D

View full text Add to dashboard Cite

The crystal structures of the azurin mutant Met l21Ala and its azide derivative Met l21Ala-azide from Pseudomonas aeruginosa have been determined. The final crystallographic R values are 21.3 and 19.4% for the two structures, respectively. In the Met l21Ala mutant, the distance between the copper ion and His117 increases by 0.34/~ compared with the wild-type structure. The removal of the methionine in the apical position induces a shortening of the distance from the copper ion to the carbonyl O atom of Gly45 from 2.97 to 2.74/~. In the Met l 21Ala-azide structure, the azide anion occupies the cavity created by replacing the Met l21 side chain with the smaller methyl group of Ala. The azide anion binds with a terminal N atom to the copper ion at a distance of about 2.04 A. In addition, the copper ion has moved out of the trigonal plane by about 0.26 A, towards the azide anion. Thus, the copper site in this structure has a distorted tetrahedral arrangement. The spectroscopic characteristics show, in addition, that the copper sites in the two structures are distinctively different. The Met l21Ala mutant still maintains the properties of an ordinary type 1 copper site while the Metl21Ala-azide derivative has an absorption maximum at about 409 nm and the copper hyperfine coupling has increased to a value intermediate between those of type 2 copper and the wild-type azurin.

show abstract

“…For an experimental validation, we tested the performance of the qTROSY recovery scheme on 4 different proteins: human ubiquitin (76 residues, Ubiquilable TM from VLI Research, www.vli-research.com); the ribonuclease barnase from Bacillus amyloliquefaciens (110 residues, BMRB 4964); the cupredoxin azurin from Pseudomonas aeruginosa in its reduced form (128 residues) (Karlsson et al, 1989); and the B1 domain of peptostreptococcal protein-L Y45W mutant, in the following called pL (64 residues) (Millet et al, 2003). All samples were [U) 15 N] labeled, pL was additionally perdeuterated.…”

Section: Resultsmentioning

confidence: 99%

qTROSY – a novel scheme for recovery of the anti-TROSY magnetisation

Diercks

Orekhov²

2005

J Biomol NMR

View full text Add to dashboard Cite

In TROSY experiments, spin state selection (S 3 ) retains only the single HSQC sub-spectrum with minimal T 2 relaxation and maximal resolution, yet at the cost of eliminating half of the available polarisation as undesired anti-TROSY component. We here introduce queued TROSY (qTROSY) as a novel scheme to partially recover and exploit this anti-TROSY polarisation in two concatenated scans. After initial orthogonal spin state separation (oS 3 ), anti-TROSY polarisation is explicitly stored while its TROSY counterpart follows the desired coherence pathway recorded in a first scan A. The immediately appended scan B then quantitatively converts the recovered anti-TROSY polarisation into a second TROSY spectrum, skipping the time-limiting long reequilibration delay. Both concatenated qTROSY scans thus ideally exploit the full initial polarisation within almost the same measurement time. In practice, T 2 relaxation losses accruing during the coupling evolution delays reduced anti-TROSY polarisation recovery below 40%, obviating sensitivity enhancement through addition of both qTROSY scans; yet, scan B retained a complete scan A spectrum with up to 75% intensity. We therefore propose to employ qTROSY asymmetrically, compacting two separate conventional into one queued TROSY-type experiment with significantly reduced measurement time, implying primarily the concatenation of different three-or higherdimensional experiments. Both anti-TROSY polarisation recoveries and possible time savings are largest for deuterated and smaller non-deuterated proteins, extending the rentability limit of the TROSY principle towards smaller molecular weights.Abbreviations: oS 3 -orthogonal spin state separation; S 3 -spin state selective; S 3 -CT -S 3 coherence transfer; S 3 E -S 3 excitation; ST2-PT -spin state to spin state selective polarisation transfer.

show abstract

Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli

Cited by 111 publications

References 17 publications

Flexibility of the metal-binding region in apo-cupredoxins

Flexibility of the metal-binding region in apo-cupredoxins

Mutant Met121Ala ofPseudomonas aeruginosaAzurin and Its Azide Derivative: Crystal Structures and Spectral Properties

qTROSY – a novel scheme for recovery of the anti-TROSY magnetisation

Contact Info

Product

Resources

About