scholarly journals The Non-Dominant AAA+ Ring in the ClpAP Protease Functions as an Anti-Stalling Motor to Accelerate Protein Unfolding and Translocation

2019 ◽  
Author(s):  
Hema Chandra Kotamarthi ◽  
Robert T. Sauer ◽  
Tania A. Baker
Keyword(s):  
Protein Unfolding ◽  
Clpap Protease

scholarly journals The non-dominant AAA+ ring in the ClpAP protease functions as an anti-stalling motor to accelerate protein unfolding and translocation

2019 ◽  
Author(s):  
Hema Chandra Kotamarthi ◽  
Robert. T. Sauer ◽  
Tania. A. Baker
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Protein Unfolding ◽  
Protein Homeostasis ◽  
Protein Substrates ◽  
Clpap Protease

AbstractATP-powered unfoldases containing D1 and D2 AAA+ rings play important roles in protein homeostasis, but uncertainty about the function of each ring remains. Here we use single-molecule optical-tweezers to assay mechanical unfolding and translocation by a variant of the ClpAP protease containing an ATPase-inactive D1 ring. This variant displays substantial mechanical defects both in unfolding and translocation of protein substrates. Notably, when D1 is hydrolytically inactive, ClpAP often stalls for times as long as minutes, and the substrate can “back-slip” through the enzyme when ATP concentrations are low. The inactive D1 variant also has substantially more difficulty traveling in the N-to-C direction on a polypeptide track than moving C-to-N. These results indicate that D1 normally functions as an auxiliary/regulatory motor to promote uninterrupted enzyme advancement that is fueled largely by the D2 ring.

scholarly journals The Non-dominant AAA+ Ring in the ClpAP Protease Functions as an Anti-stalling Motor to Accelerate Protein Unfolding and Translocation

Cell Reports ◽  
2020 ◽  
Vol 30 (8) ◽  
pp. 2644-2654.e3 ◽  
Author(s):  
Hema Chandra Kotamarthi ◽  
Robert T. Sauer ◽  
Tania A. Baker
Keyword(s):  
Protein Unfolding ◽  
Clpap Protease

scholarly journals Protein unfolding by SDS: the microscopic mechanisms and the properties of the SDS-protein assembly

Nanoscale ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 5422-5434 ◽  
Author(s):  
David Winogradoff ◽  
Shalini John ◽  
Aleksei Aksimentiev
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Anionic Surfactant ◽  
Protein Unfolding ◽  
Protein Assembly ◽  
Full Length ◽  
Dynamics Simulations

Molecular dynamics simulations reveal how anionic surfactant SDS and heat unfold full-length proteins.

Evidence of an Intermediate and Parallel Pathways in Protein Unfolding from Single-Molecule Fluorescence

2008 ◽  
Vol 130 (25) ◽  
pp. 7898-7907 ◽  
Author(s):  
Angel Orte ◽  
Timothy D. Craggs ◽  
Samuel S. White ◽  
Sophie E. Jackson ◽  
David Klenerman
Keyword(s):  
Single Molecule ◽  
Protein Unfolding ◽  
Single Molecule Fluorescence ◽  
Parallel Pathways

scholarly journals Universal Screening Methods and Applications of ThermoFluor®

2006 ◽  
Vol 11 (7) ◽  
pp. 854-863 ◽  
Author(s):  
Maxwell D. Cummings ◽  
Michael A. Farnum ◽  
Marina I. Nelen
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Protein Unfolding ◽  
Direct Detection ◽  
Functional Characterization ◽  
Screening Methods ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Bacterial Enzyme ◽  
Research Problems

The genomics revolution has unveiled a wealth of poorly characterized proteins. Scientists are often able to produce milligram quantities of proteins for which function is unknown or hypothetical, based only on very distant sequence homology. Broadly applicable tools for functional characterization are essential to the illumination of these orphan proteins. An additional challenge is the direct detection of inhibitors of protein-protein interactions (and allosteric effectors). Both of these research problems are relevant to, among other things, the challenge of finding and validating new protein targets for drug action. Screening collections of small molecules has long been used in the pharmaceutical industry as 1 method of discovering drug leads. Screening in this context typically involves a function-based assay. Given a sufficient quantity of a protein of interest, significant effort may still be required for functional characterization, assay development, and assay configuration for screening. Increasingly, techniques are being reported that facilitate screening for specific ligands for a protein of unknown function. Such techniques also allow for function-independent screening with better characterized proteins. ThermoFluor®, a screening instrument based on monitoring ligand effects on temperature-dependent protein unfolding, can be applied when protein function is unknown. This technology has proven useful in the decryption of an essential bacterial enzyme and in the discovery of a series of inhibitors of a cancer-related, protein-protein interaction. The authors review some of the tools relevant to these research problems in drug discovery, and describe our experiences with 2 different proteins.

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