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Structure of proteins in eukaryotic compartments.

ABSTRACT: In-cell NMR in the yeast Pichia pastoris was used to study the influence of metabolic changes on protein structure and dynamics at atomic resolution. Induction of ubiquitin overexpression from the methanol induced AOX1 promoter results in the protein being localized in the cytosol and yields a well-resolved in-cell NMR spectrum. When P. pastoris is grown on a mixed carbon source containing both dextrose and methanol, ubiquitin is found in small storage vesicles distributed in the cytosol, and the resulting in-cell NMR spectrum is broadened. The sequestration of overexpressed proteins into storage vesicles, which are inaccessible to small molecules, was demonstrated for two unrelated proteins and two different strains of P. pastoris , suggesting its general nature.

SUBMITTER: Bertrand K

PROVIDER: S-EPMC3417295 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Structure of proteins in eukaryotic compartments.

Bertrand Karl K Reverdatto Sergey S Burz David S DS Zitomer Richard R Shekhtman Alexander A

Journal of the American Chemical Society 20120719 30

In-cell NMR in the yeast Pichia pastoris was used to study the influence of metabolic changes on protein structure and dynamics at atomic resolution. Induction of ubiquitin overexpression from the methanol induced AOX1 promoter results in the protein being localized in the cytosol and yields a well-resolved in-cell NMR spectrum. When P. pastoris is grown on a mixed carbon source containing both dextrose and methanol, ubiquitin is found in small storage vesicles distributed in the cytosol, an ...[more]

PMID: 22758659

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Project description:A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.

Dataset Information

Structure of proteins in eukaryotic compartments.

Publications

Structure of proteins in eukaryotic compartments.

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