In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Protein quality control in the endoplasmic reticulum is of central importance for cellular homeostasis in eukaryotes. Crucial for this process is the HRD-ubiquitin ligase (HMG-CoA reductase degradation), which singles out terminally misfolded proteins and routes them for degradation to cytoplasmic 26S-proteasomes. Certain functions of this enzyme complex are allocated to defined subunits. However, it remains unclear how these components act in a concerted manner. Here, we show that Usa1 functions as a major scaffold protein of the HRD-ligase. For the turnover of soluble substrates, Der1 binding to the C terminus of Usa1 is required. The N terminus of Usa1 associates with Hrd1 and thus bridges Der1 to Hrd1. Strikingly, the Usa1 N terminus also induces oligomerization of the HRD complex, which is an exclusive prerequisite for the degradation of membrane proteins. Our data demonstrate that scaffold proteins are required to adapt ubiquitin ligase activities toward different classes of substrates.

The RNA-binding protein Lin28 regulates the processing of a developmentally important group of microRNAs, the let-7 family. Lin28 blocks the biogenesis of let-7 in embryonic stem cells and thereby prevents differentiation. It was shown that both RNA-binding domains (RBDs) of this protein, the cold-shock domain (CSD) and the zinc-knuckle domain (ZKD) are indispensable for pri- or pre-let-7 binding and blocking its maturation. Here, we systematically examined the nucleic acid-binding preferences of the Lin28 RBDs and determined the crystal structure of the Lin28 CSD in the absence and presence of nucleic acids. Both RNA-binding domains bind to single-stranded nucleic acids with the ZKD mediating specific binding to a conserved GGAG motif and the CSD showing only limited sequence specificity. However, only the isolated Lin28 CSD, but not the ZKD, can bind with a reasonable affinity to pre-let-7 and thus is able to remodel the terminal loop of pre-let-7 including the Dicer cleavage site. Further mutagenesis studies reveal that the Lin28 CSD induces a conformational change in the terminal loop of pre-let-7 and thereby facilitates a subsequent specific binding of the Lin28 ZKD to the conserved GGAG motif.

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 Å resolution and refined to a crystallographic R-factor of 20.5% (R free 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open a/b topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20°in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.Keywords: crystal structure; protein crystallography; pyruvate decarboxylase; substrate specificity; thiamin diphosphate.Plant hormones play central roles in the regulation of plant growth and development. The first plant hormone to be described was indole-3-acetic acid (IAA), which is synthesized by plants [1,2] and plant-associated bacteria [3,4]. Several pathways for the synthesis of IAA in these organisms have been described, and most of them start from L-tryptophan as precursor. One of the tryptophandependent biosynthetic routes to IAA is the indolepyruvic acid (IPA) pathway. This pathway starts from L-tryptophan, and consists of three steps: (a) the conversion of tryptophan to indole-3-pyruvic acid; (b) the formation of indole-3-acetaldehyde; and (c) the production of IAA (Fig. 1). The first step of the pathway is catalysed by L-tryptophan aminotransferase, a pyridoxal-5-phosphatedependent enzyme [5]. The intermediate, IPA, is decarboxylated by the action of indolepyruvate decarboxylase (IPDC) [6] and the resulting indole-3-acetaldehyde is oxidized by an aldehyde oxidase to IAA [7].Genes encoding IPDC from several microorganisms have been cloned and characterized. These organisms include Enterobacter cloacae [8], Pantoea agglomerans [9], Klebsiella aerogenes [10], Azospirillum brasilense [11,12] and Azospirillum lipoferum [13]. The IPDC genes code for polypeptides of about 550 amino acids in length, corresponding to a molecular mass of 60 kDa per subunit. The enzyme from E. cloacae, which has been characterized biochemically to some extent, has a molecular mass of 240 kDa, suggesting a...

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole‐3‐acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole‐3‐acetic acid, catalyses the formation of indole‐3‐acetaldehyde and carbon dioxide from indole‐3‐pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole‐3‐pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped‐flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole‐3‐pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X‐ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH‐dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207–1216] and comparison with indole‐3‐pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.