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Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae
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Published on: July 9, 2013

Preferential substrate binding orientation by the molecular chaperone HscA.

Tim L Tapley1, Larry E Vickery

  • 1Department of Physiology and Biophysics, University of California-Irvine, Irvine, CA 92697, USA.

The Journal of Biological Chemistry
|April 22, 2004
PubMed
Summary

This study investigated how a bacterial chaperone called HscA binds to a protein involved in iron-sulfur cluster assembly. Researchers used fluorescence labeling to determine whether HscA interacts with the target protein in a specific orientation. They found that HscA binds in a directional manner, with the orientation influenced by the position of a specific amino acid in the target protein. The findings suggest that HscA and other hsp70 chaperones may recognize substrates differently, depending on their isoform. This could help explain how chaperones assist in protein folding and assembly processes.

Keywords:
HscA bindingIscU interactionhsp70 substrate recognitionchaperone orientationprotein folding mechanism

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Published on: July 21, 2021

Area of Science:

  • Molecular chaperone function in protein folding
  • Structural biology of hsp70-class chaperones
  • Iron-sulfur cluster assembly mechanisms

Background:

Hsp70-class chaperones are known to assist in protein folding and assembly. Prior research has shown that these proteins interact with substrates through conserved motifs. However, the orientation of substrate binding remains unclear in many cases. Some studies suggest that binding orientation may differ among hsp70 isoforms. No prior work had resolved whether HscA binds substrates in a specific orientation. This gap motivated a detailed investigation into HscA's interaction with IscU. The LPPVK motif is a key feature of IscU that may influence binding. Understanding binding orientation could clarify how HscA contributes to iron-sulfur cluster assembly.

Purpose Of The Study:

The study aimed to determine whether HscA binds IscU in a preferred orientation. Researchers focused on the LPPVK motif and its role in substrate recognition. They used fluorescence labeling and quenching to assess binding orientation. The goal was to compare peptide and full-length IscU binding. The study also examined the effect of HscB on binding orientation. Prior work suggested variability in hsp70 binding orientation. This study sought to confirm whether HscA-IscU binding follows a distinct pattern. The findings could clarify how HscA functions in iron-sulfur cluster assembly.

Main Methods:

Researchers used site-directed fluorescence labeling with bimane to study HscA binding. Fluorescent probes were attached to opposite sides of the HscA domain. Peptides containing LPPVK and tryptophan at either terminus were tested. Fluorescence quenching was measured to assess binding orientation. The position of tryptophan in the peptide influenced quenching efficiency. Experiments compared peptide binding to full-length IscU binding. HscB's effect on binding orientation was also evaluated. The study combined fluorescence spectroscopy with structural analysis.

Main Results:

Fluorescence quenching varied significantly based on tryptophan position and bimane location. Peptides with tryptophan at the N terminus showed stronger quenching than those at the C terminus. This suggests a directional preference for HscA binding. Full-length IscU bound in the same orientation as its derived peptides. HscB did not alter the binding orientation of HscA-IscU. The preferred orientation of HscA-IscU is opposite to that of DnaK and rat Hsc70. This finding indicates variability among hsp70 isoforms in binding orientation. The results support a model where HscA interacts with IscU in a specific, conserved manner.

Conclusions:

The study supports the authors' claim that HscA binds IscU in a preferred orientation. Fluorescence quenching data indicate a directional preference for peptide binding. Full-length IscU binds in the same orientation as its derived peptides. HscB does not influence binding orientation, according to the authors. The preferred orientation of HscA-IscU is opposite to that of DnaK and rat Hsc70. This suggests that hsp70 isoforms may recognize substrates differently. The findings clarify how HscA contributes to iron-sulfur cluster assembly. The authors propose that binding orientation is conserved within the HscA-IscU complex.

Fluorescence quenching experiments suggest that HscA binds IscU in a directional manner. Tryptophan position in peptides influences binding orientation.

The LPPVK motif is a conserved sequence in IscU that HscA recognizes. Peptides containing this motif were used to study binding orientation.

Tryptophan at the N terminus of peptides caused stronger quenching than at the C terminus. This suggests a directional preference for HscA binding.

HscA-IscU binding orientation is the reverse of that seen in DnaK and rat Hsc70. This indicates variability among hsp70 isoforms.

Experiments showed that HscB does not influence the binding orientation of HscA-IscU. The orientation remains unchanged.

The authors propose that hsp70 isoforms may bind substrates in different orientations. This could explain functional differences among chaperones.