Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s), whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.
Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs) containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1) domains, while the membrane-distal (D2) domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.
Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of threephase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.Keywords: three-phase partitioning; protein refolding; recombinant ribonuclease A; CcdB mutants; human CD4; inclusion bodies Supplemental material: see www.proteinscience.org Upon expression in Escherichia coli, many recombinant proteins form dense, insoluble aggregates called inclusion bodies (Taylor et al. 1986). It is therefore necessary to develop efficient and scalable procedures for solubilization and refolding of recombinant proteins from inclusion bodies (Misawa and Kumagai 1999;Middelberg 2002).Refolding is typically carried out using either simple dilution into appropriate refolding conditions, dialysis or on a column (Stempfer et al. 1996;Rogl et al. 1998). Finding appropriate refolding conditions is generally the most difficult step of the purification process. Aggregation and precipitation of protein during refolding are commonly encountered problems. Factorial and other screens for refolding have been developed (Hofmann et al. 1995;Armstrong et al. 1999;Bajaj et al. 2004) to facilitate the search of appropriate refolding conditions. In the present study we describe the first application of the technique of three-phase partitioning (TPP) (Lovrien et al. 1995;Dennison and Lovrien 1997;Jain et al. 2004;Przybycien et al. 2004) to obtain active refolded proteins from solubilized inclusion bodies without any chromatographic steps. We have used TPP to refold 12 different proteins from inclusion bodies, namely, ribonuclease A Reprint requests to: Munishwar N. Gupta, Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India; e-mail: munishwar48@yahoo.co.uk; fax: 91-011-26581073.Abbreviations: CcdB, controller of cell division or death B; CD4D12, first two domains of human CD4; C m , the denaturant concentration at which one half of the protein molecules are unfolded; GdmCl, guanidium chloride; IPTG, isopropyl b-D-1-thiogalactopyranoside; MBP, maltose binding protein; PTPs, protein tyrosine phosphatases; RNase A, ribonuclease A; SPR, surface plasmon resonance; TPP, three-phase partitioning; Trx, E. coli thioredoxin.Article and publication are at
The production of recombinant proteins in Escherichia coli involves substantial optimization in the size of the protein and over-expression strategies to avoid inclusion-body formation. Here we report our observations on this so-called construct dependence using the catalytic domains of five Drosophila melanogaster receptor protein tyrosine phosphatases as a model system. Five strains of E. coli as well as three variations in purification tags viz., poly-histidine peptide attachments at the N- and C-termini and a construct with Glutathione-S-transferase at the N-terminus were examined. In this study we observe that inclusion of a 45 residue stretch at the N-terminus was crucial for over-expression of the enzymes, influencing both the solubility and the stability of these recombinant proteins. While the addition of negatively charged residues in the N-terminal extension could partially rationalize the improvement in the solubility of these constructs, conventional parameters like the proportion of order promoting residues or aliphatic index did not correlate with the improved biochemical characteristics. These findings thus suggest the inclusion of additional parameters apart from rigid domain predictions to obtain domain constructs that are most likely to yield soluble protein upon expression in E. coli.
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