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Title: Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements

Abstract

Final Project Report describing work to elucidate mechanisms for the activation of [FeFe]-hydrogenases and to explore the impact of the polypeptide scaffolding on the function of the Fe-S redox and catalytic centers with emphasis on improving oxygen tolerance.

Authors:
 [1]
  1. Stanford Univ., CA (United States)
Publication Date:
Research Org.:
Stanford Univ., CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1341627
DOE Contract Number:
Sc0002010
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Hydrogen; Hydrogenase; Photosynthetic

Citation Formats

Swartz, James. Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements. United States: N. p., 2017. Web. doi:10.2172/1341627.
Swartz, James. Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements. United States. doi:10.2172/1341627.
Swartz, James. Wed . "Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements". United States. doi:10.2172/1341627. https://www.osti.gov/servlets/purl/1341627.
@article{osti_1341627,
title = {Using in vitro maturation and cell-free expression to explore [FeFe] hydrogenase activation and protein scaffolding requirements},
author = {Swartz, James},
abstractNote = {Final Project Report describing work to elucidate mechanisms for the activation of [FeFe]-hydrogenases and to explore the impact of the polypeptide scaffolding on the function of the Fe-S redox and catalytic centers with emphasis on improving oxygen tolerance.},
doi = {10.2172/1341627},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed Jan 25 00:00:00 EST 2017},
month = {Wed Jan 25 00:00:00 EST 2017}
}

Technical Report:

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  • The in vitro activation of the [FeFe] hydrogenase is accomplished by combining Escherichia coli cell extracts containing the heterologously expressed inactive HydA with extracts in which hydrogenase-specific maturation proteins HydE, HydF, and HydG are expressed in concert. Interestingly, the process of HydA activation occurs rapidly and in the absence of potential substrates, which suggests that the hydrogenase accessory proteins synthesize an H-cluster precursor that can be quickly transferred to the hydrogenase enzyme to affect activation. HydA activity is observed to be dependent on the protein fraction containing all three accessory proteins expressed in concert and cannot be accomplished with additionmore » of heat-treated extract or extract filtrate, suggesting that the activation of the hydrogenase structural protein is mediated by interaction with the accessory assembly protein(s). These results represent the first important step in understanding the process of H-cluster assembly and provide significant insights into hydrogenase maturation.« less
  • Experiments examined the effects of radiation dose-rate and protein synthesis inhibition expression of cytoskeletal and matrix elements in Syrian hamster embryo cells. Results demonstrated little effect of dose-rate for neutrons when comparing expression of {alpha}-tubulin and fibronectin genes. Cycloheximide repressed accumulation of {alpha}-tubulin-mRNA following exposure to high dose-rate neutrons or {gamma} rays. Cycloheximide did not affect accumulation of actin mRNA. Cycloheximide abrogated induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to radiation. 24 refs., 3more » tabs.« less
  • Experiments were designed to examine the effects of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Results demonstrated little effect of dose-rate for JANUS fission-spectrum neutrons when comparing expression of either a-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Cycloheximide, however, repressed accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposures. Cycloheximide did not affect accumulation of mRNA for actinmore » genes. Cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin mRNA accumulation following exposure to ionizing radiation and that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.« less
  • Experiments were designed to examine the effects Of radiation dose-rate and of the protein synthesis inhibitor cycloheximide on expression of cytoskeletal elements ({gamma}- and {beta}-actin and {alpha}-tubulin) and matrix elements (fibronectin) in Syrian hamster embryo cells. Past work from our laboratory had already demonstrated optimum time points and doses for examination of radiation effects on accumulation of specific transcripts. Our results here demonstrated little effect of dose-rate for JANUS fission spectrum neutrons when comparing expression of either {alpha}-tubulin or fibronectin genes. Past work had already documented similar results for expression of actin transcripts. Effects of cycloheximide revealed that cycloheximide repressedmore » accumulation of {alpha}-tubulin following exposure to high dose-rate neutrons or {gamma} rays; this did not occur following similar low dose-rate exposure. (2) Cycloheximide did not affect accumulation of MRNA for actin genes; and that cycloheximide abrogated the moderate induction of fibronectin-mRNA which occurred following exposure to {gamma} rays and high dose-rate neutrons. These results suggest a role for labile proteins in the maintenance of {alpha}-tubulin and fibronectin MRNA accumulation following exposure to ionizing radiation. in addition, they suggest that the cellular/molecular response to low dose-rate neutrons may be different from the response to high dose-rate neutrons.« less