Global analysis of protein structural changes in complex proteomes

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Abstract

Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for 300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.

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Figure 1: LiP-SRM workflow.
Figure 2: LiP-SRM of α-Syn and myoglobin spiked into complex cell extracts.
Figure 3: Global analysis of protein conformational changes.
Figure 4: LiP-SRM analysis of core carbon metabolism upon a shift from glycolytic to gluconeogenic growth.
Figure 5: Structural changes of Cdc19 upon a switch from glucose- to ethanol-based metabolism.
Figure 6: Structural transition of the yeast 14-3-3 protein Bmh1 upon a switch from glucose- to ethanol-based metabolism.

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Acknowledgements

P.P. is supported by a 'Foerderungsprofessur' grant from the Swiss National Science Foundation (grant PP00P3_133670), by an EU Seventh Framework Program Reintegration grant (FP7-PEOPLE-2010-RG-277147) and by a Promedica Stiftung (grant 2-70669-11). Y.F. is supported by an ETH Research Grant (grant 4412-1); M.S. is supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship D award. G.D.F. is supported by a post-doctoral fellowship of the University of Padua. A.P.O. is supported by the SystemsX.ch project YeastX. We thank R. Costenoble, K. Kochanowski and U. Sauer (ETH Zurich) for insightful discussions and for the measurements of the intracellular concentrations of FBP and M. Peter for access to plasmid and strain collections. We are grateful to O. Vitek and M. Choi (Purdue University), R. Riek, C. Chi and P. Navarro (ETH Zurich) for helpful discussions. We also thank P. Nanni and R. Schlapbach from the Functional Genomics Centre Zurich for access to mass spectrometry instrumentation, F. Allain for access to the D-BIOL Biomolecular NMR Spectroscopy Platform at the ETH Zurich, N. Ban and M.A. Leibundgut for providing a sample of purified yeast fatty acid synthase.

Author information

P.P. conceived and supervised the project. Y.F., G.D.F. and P.P. designed and performed the experiments. Y.F., G.D.F., M.S. and A.M. performed experiments and analyzed the data. P.B. contributed to mass spectrometry measurements. P.P.d.L. supervised parts of the project. A.K. analyzed the data. A.P.O. and Y.N. contributed to the analysis and validation of the metabolic data. P.P., Y.F., G.D.F. and A.K. wrote the manuscript.

Correspondence to Paola Picotti.

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Competing interests

P.P., Y.F. and G.D.F. are inventors on a patent application that pertains to the method presented in this study.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 2–6 and Supplementary Note (PDF 4522 kb)

Supplementary Table 1

Complete set of SRM assays used in this study (XLSX 514 kb)

Supplementary Table 7

LiP sites identified from spectral count cata (XLSX 664 kb)

Supplementary Table 8

Structural properties of proteins subject to LiP cleavage in the proteome of yeast grown in glucose-based medium (XLSX 628 kb)

Supplementary Table 9

Proteins that change conformational properties upon transition from glucose to ethanol growth conditions (XLSX 81 kb)

Supplementary Table 10

Background and target proteomes used in the functional enrichment analysis (XLSX 64 kb)

Supplementary Table 11

Changes in abundance and in the LiP pattern for metabolic enzymes for yeast grown in ethanol, relative to yeast grown in glucose (XLSX 21 kb)

Supplementary Table 12

Changes in the LiP pattern of TCA cycle enzymes (XLSX 43 kb)

Supplementary Table 13

Changes in the LiP pattern of pyruvate kinase (XLSX 15 kb)

Supplementary Table 14

Reversion of the proteolytic pattern of Cdc19 by addition of FBP to the extract from cells grown in ethanol (XLSX 14 kb)

Supplementary Table 15

Changes in LiP pattern of proteins in the proteome of yeast grown in ethanol upon administration of FBP (XLSX 44 kb)

Supplementary Table 16

Changes in the LiP pattern of fatty acid synthase subunits 1 and 2 (Fas1 and Fas2) upon FBP administration (XLSX 82 kb)

Supplementary Table 17

Changes in the LiP pattern of Bmh1 and Bmh2 (XLSX 13 kb)

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Feng, Y., De Franceschi, G., Kahraman, A. et al. Global analysis of protein structural changes in complex proteomes. Nat Biotechnol 32, 1036–1044 (2014). https://doi.org/10.1038/nbt.2999

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