Edited by Stuart FergusonKeywords: Domain movement Substrate and metal-effect 3-Isopropylmalate dehydrogenase Small-angle X-ray scattering a b s t r a c t X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal-IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0-5%) extent of domain closure in the absence of the metal-ion. Only the metal-IPM complex, but neither the metal-ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal-effect is given.
Edited by Peter Brzezinski Keywords:Isopropylmalate dehydrogenase Activation by K + Site-directed mutagenesis X-ray crystallography Small angle X-ray scattering Fluorescence resonance energy transfer a b s t r a c tThe mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced ($1%) catalytic activity and negligible activation by K + compared to the wild-type enzyme. A 3-4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K + . SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K + into close proximity of the nicotinamide ring of NAD + and the electronwithdrawing effect of K + may help to polarise the aromatic ring in order to aid the hydride-transfer.
The tumour suppressor protein RASSF1A is phosphorylated by Aurora A kinase, thereby impairing its tumour suppressor function. Consequently, inhibiting the interaction between Aurora A and RASSF1A may be used for anti-tumour therapy. We used recombinant variants of RASSF1A to map the sites of interaction with Aurora A. The phosphorylation kinetics of three truncated RASSF1A variants has been analysed. Compared to the RASSF1A form lacking the 120 residue long N-terminal part, the K m value of the phosphorylation is increased from 10 to 45 μM upon additional deletion of the C-terminal SARAH domain. On the other hand, deletion of the flexible loop (Δ177–197) that precedes the phosphorylation site/s (T202/S203) results in a reduction of the k cat value from about 40 to 7 min −1 . Direct physical interaction between the isolated SARAH domain and Aurora A was revealed by SPR. These data demonstrate that the SARAH domain of RASSF1A is involved in the binding to Aurora A kinase. Structural modelling confirms that a novel complex is feasible between the SARAH domain and the kinase domain of Aurora A. In addition, a regulatory role of the loop in the catalytic phosphorylation reaction has been demonstrated both experimentally and by structural modelling.
We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.