Nucleotide Exchange Factors for Hsp70 Chaperones
Heike Rampelt1,2, Matthias P Mayer3, Bernd Bukau1
1Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|November 28, 2017
Summary
This study details methods for analyzing nucleotide exchange factors (NEFs) that regulate Hsp70 chaperone protein folding. Assays measure how NEFs affect the Hsp70 ATPase cycle and nucleotide release, aiding in NEF identification.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Chaperone Proteins
Background:
- Hsp70 chaperones are crucial for protein folding, with their function dictated by their ATPase cycle.
- Nucleotide exchange factors (NEFs) modulate Hsp70 activity by accelerating ADP release, impacting substrate binding and release dynamics.
Purpose of the Study:
- To describe methodologies for the study and characterization of Hsp70 nucleotide exchange factors (NEFs).
- To provide tools for understanding NEF influence on Hsp70 ATPase activity and substrate interactions.
Main Methods:
- Steady-state ATPase assays to evaluate NEF impact on the Hsp70 ATPase cycle.
- Direct measurement of nucleotide release using labeled nucleotides for NEF identification and characterization.
Main Results:
- Steady-state ATPase assays reveal NEF-mediated alterations in Hsp70's ATPase cycle progression.
- Direct nucleotide release assays enable the identification and detailed characterization of NEFs.
Conclusions:
- The described methods are suitable for studying NEFs and their role in Hsp70 chaperone function.
- These techniques facilitate a deeper understanding of the regulation of Hsp70-substrate complex lifetime.
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