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FP5

SAMBA Sintesi della relazione

Project ID: IST-2000-29685
Finanziato nell'ambito di: FP5-IST
Paese: Netherlands

Production and characterization of ET proteins

The modification, production and characterization of variants of a series of blue copper proteins and redox enzymes was undertaken. Cu proteins were produced in the native form, the apo-form, and the Zn-containing form. The proteins were tailored to user specifications as the project developed. New variants could be offered as functional characterisation of the protein and enzyme variants progressed in Leiden. In addition clones of protein variants were provided on demand. The mutations applied to the various proteins and enzymes pertained to the engineering of surface cysteines, allowing one-, two- or three-point immobilizations at solid surfaces, and the creation of cavity mutants allowing the use of hot wires. Additional work related to the synthesis of hot wires and the construction of specialised clones. The design of new variants was routinely based on extensive modeling.

Construction at the DNA-level was achieved by state-of the-art rec-DNA techniques. Expression and purification of the proteins/enzymes was achieved according to advanced protein chemistry techniques. Purification protocols were developed or adjusted where necessary. Purification protocols for the Cys-containing variants were developed with a special eye on keeping the engineered cysteine(s) active for immobilization on solid substrates.

Characterisation of the proteins/enzymes was achieved by standard biochemical techniques. Spectroscopic characterisation involved UV-VIS spectroscopy, mass spec analysis, protein film voltammetry, cyclic voltammetry and, when necessary, EPR and NMR spectroscopy. Activity was checked by enzymological and kinetic techniques (stopped flow, NMR). At a later stage of the project proteins/enzymes were also characterised for their behaviour on solid supports (gold, mica, carbon) by means of atomic force microscopy (AFM) both under ambient conditions and in situ. Variants were prepared of azurin from Pseudomonas aeruginosa, of pseudo-azurin from Alcaligenes faecalis S-6, of plastocyanin from Populus nigra and of amicyanin from P. versutus. The enzymes Nitrite reductase (NiR) from Alcaligenes faecalis S-6 and Methylamine dehydrogenase from Paracoccus versutus were produced and NiR-variants were constructed and produced that contained cysteines at various positions. In addition Heme-cd1-containing NiR and Nitric oxide reductase were made available. Details are provided in the overview below.

Overview of Metallo-proteins, wt and variants produced for SAMBA:

ET Metallo-proteins: wild types & variants

Azurin [Az, small cupredoxin]: wild type forms & variants
-- Wild type forms (Cu, Zn, apo)
-- Single surface cysteine: Q12C, K27C (Cu, Zn, apo),
N42C, S118C
-- Click-on chemistry cavity mutant H117G
-- Removal S-S bridges: C3AC26A

Plastocyanin[Pc, small cupredoxin]: wild type forms & variants
-- Engineered S-S bridge: I21C, E25C (PCSS)
-- C-terminal extension TCG (Thr-Cys-Gly) (PCSH)

Pseudoazurin [PsAzu, small cupredoxin]: wild type forms & variants
-- Single & Double surface cysteines: E51C, E54C,
-- E51CE54C

Amicyanin [Ami, small cupredoxin]: wild type forms

Enzymes: wild types & variants

Copper-containing nitrite reductase [NiR, multicopper oxidase]: wild type forms & variants:
-- Type-1 site depleted, Type-2 site depleted
-- Single surface cysteine: L93C, M94C
-- Click-on chemistry cavity H145A/G, M150G, H306A

Heme/cd1 containing nitrite reductase [cd1NiR, multi heme enzyme] :wild type

Nitric oxide reductase [Nor, multi heme enzyme] :wild type

Methylamine dehydrogenase [MADH ] : wild type

Others:
Azurin antibodies
Hotwire linker (1-(11-mercapto-undecyl)-imidazole dimer)
Plasmid containing the gene of azurin.

Contatto

Gerard W. CANTERS, (Chair of Biophysical Chemistry)
Tel.: +31-715274256
Fax: +31-715274349
E-mail