Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli - Publication - Bridge of Knowledge

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Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli

Abstract

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl cis/trans isomerases (PPIs). The Escherichia coli cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed. Measurement of their PPIase activity revealed that PpiB is the major source of PPIase activity in the cytoplasm. Furthermore, viable Δ6ppi strains could be constructed only on minimal medium in the temperature range of 30–37 °C, but not on rich medium. To address the molecular basis of essentiality of PPIs, proteins that aggregate in their absence were identified. Next, wild-type and putative active site variants of FkpB, FklB, PpiB and PpiC were purified and in pull-down experiments substrates specific to each of these PPIs identified, revealing an overlap of some substrates. Substrates of PpiC were validated by immunoprecipitations using extracts from wild-type and PpiC-H81A strains carrying a 3xFLAG-tag appended to the C-terminal end of the ppiC gene on the chromosome. Using isothermal titration calorimetry, RpoE, RseA, S2, and AhpC were established as FkpB substrates and PpiC’s PPIase activity was shown to be required for interaction with AhpC.

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Category:
Articles
Type:
artykuły w czasopismach
Published in:
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES no. 21,
ISSN: 1661-6596
Language:
English
Publication year:
2020
Bibliographic description:
Klein-Raina G., Wojtkiewicz P., Biernacka D., Stupak A., Gorzelak P., Raina S.: Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli// INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES -Vol. 21,iss. 12 (2020), s.4212-
DOI:
Digital Object Identifier (open in new tab) 10.3390/ijms21124212
Sources of funding:
Verified by:
Gdańsk University of Technology

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