Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site.

Histidine (His) tags are one of the most popular fusion tags for the isolation of proteins via metal affinity chromatography. The fusion tag is routinely left attached to the protein when carrying out experiments, with the assumption that the addition has no effect on structure or function. In the present study, we have prepared four proteins of the gene regulatory protein AreA from Aspergillus nidulans for crystallization experiments: a 91-amino acid peptide encompassing the minimal DNA-binding region, both with and without the His-tag (HZFB and ZFB, respectively), and a 155-amino acid protein previously proposed to be the entire DNA-binding domain for AreA, both with and without the His-tag (HG1b and G1b, respectively).

Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture.

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp.

A de novo redesign of the WW domain.

We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small beta-sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS-WW1 and SPANS-WW2), using only naturally occurring L-amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli.