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Structural Basis for Modulation of Quality Control Fate in a Marginally Stable Protein.

Brock KP, Abraham AC, Amen T, Kaganovich D, England JL - Structure (2015)

Bottom Line: We combined computational methods with in vivo experiments to examine the basis of the misfolding propensity of VHL.Subsequent modeling suggested the mutation had caused a conformational change affecting cofactor and chaperone interaction, and this hypothesis was then confirmed by additional knockout and overexpression experiments targeting a yeast cofactor homolog.These findings offer a detailed structural basis for the modulation of quality control fate in a model misfolded protein and highlight burial mode modeling as a rapid means to detect functionally important conformational changes in marginally stable globular domains.

View Article: PubMed Central - PubMed

Affiliation: Computational and Systems Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

No MeSH data available.


Related in: MedlinePlus

Analysis of VHL Mutant Stability by Flow Cytometry and Fluorescence Microscopy(A) Fluorescence measurements by quantitative flow cytometry for all experimentally created VHL mutations and normalized to fluorescence of the wild-type sequence. The orange entry indicates the most stable mutant, VHL19 (L201-E173), in both panels.(B) Selected mutants were also introduced to S. cerevisiae cells and expressed endogenously as a GFP fusion construct, and observed directly by fluorescence microscopy with results normalized to GFP levels without VHL. Error bars for all sections represent SE. See also degradation curves in Figure S1. a.u., arbitrary units.
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fig2: Analysis of VHL Mutant Stability by Flow Cytometry and Fluorescence Microscopy(A) Fluorescence measurements by quantitative flow cytometry for all experimentally created VHL mutations and normalized to fluorescence of the wild-type sequence. The orange entry indicates the most stable mutant, VHL19 (L201-E173), in both panels.(B) Selected mutants were also introduced to S. cerevisiae cells and expressed endogenously as a GFP fusion construct, and observed directly by fluorescence microscopy with results normalized to GFP levels without VHL. Error bars for all sections represent SE. See also degradation curves in Figure S1. a.u., arbitrary units.

Mentions: All 20 mutations were then created in an S. cerevisiae constitutive plasmid with an attached fluorescent Dendra2 tag and transformed into budding yeast cells, which contain neither native VHL protein nor the human elongin cofactors. If the VHL protein were able to adopt a fold that is resistant to PQC degradation, then the attached fluorescent tag would also be preserved and observable. Fluorescence was detected by using quantitative flow cytometry (Figure 2A). A subset of these mutants were then tagged with GFP and integrated into the genome under the control of a galactose-operated promoter, and their expression level was quantified by fluorescence microscopy at steady-state levels (Figure 2B).


Structural Basis for Modulation of Quality Control Fate in a Marginally Stable Protein.

Brock KP, Abraham AC, Amen T, Kaganovich D, England JL - Structure (2015)

Analysis of VHL Mutant Stability by Flow Cytometry and Fluorescence Microscopy(A) Fluorescence measurements by quantitative flow cytometry for all experimentally created VHL mutations and normalized to fluorescence of the wild-type sequence. The orange entry indicates the most stable mutant, VHL19 (L201-E173), in both panels.(B) Selected mutants were also introduced to S. cerevisiae cells and expressed endogenously as a GFP fusion construct, and observed directly by fluorescence microscopy with results normalized to GFP levels without VHL. Error bars for all sections represent SE. See also degradation curves in Figure S1. a.u., arbitrary units.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4509718&req=5

fig2: Analysis of VHL Mutant Stability by Flow Cytometry and Fluorescence Microscopy(A) Fluorescence measurements by quantitative flow cytometry for all experimentally created VHL mutations and normalized to fluorescence of the wild-type sequence. The orange entry indicates the most stable mutant, VHL19 (L201-E173), in both panels.(B) Selected mutants were also introduced to S. cerevisiae cells and expressed endogenously as a GFP fusion construct, and observed directly by fluorescence microscopy with results normalized to GFP levels without VHL. Error bars for all sections represent SE. See also degradation curves in Figure S1. a.u., arbitrary units.
Mentions: All 20 mutations were then created in an S. cerevisiae constitutive plasmid with an attached fluorescent Dendra2 tag and transformed into budding yeast cells, which contain neither native VHL protein nor the human elongin cofactors. If the VHL protein were able to adopt a fold that is resistant to PQC degradation, then the attached fluorescent tag would also be preserved and observable. Fluorescence was detected by using quantitative flow cytometry (Figure 2A). A subset of these mutants were then tagged with GFP and integrated into the genome under the control of a galactose-operated promoter, and their expression level was quantified by fluorescence microscopy at steady-state levels (Figure 2B).

Bottom Line: We combined computational methods with in vivo experiments to examine the basis of the misfolding propensity of VHL.Subsequent modeling suggested the mutation had caused a conformational change affecting cofactor and chaperone interaction, and this hypothesis was then confirmed by additional knockout and overexpression experiments targeting a yeast cofactor homolog.These findings offer a detailed structural basis for the modulation of quality control fate in a model misfolded protein and highlight burial mode modeling as a rapid means to detect functionally important conformational changes in marginally stable globular domains.

View Article: PubMed Central - PubMed

Affiliation: Computational and Systems Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

No MeSH data available.


Related in: MedlinePlus