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Cyanylated Cysteine Reports Site-Specific Changes at Protein–Protein-Binding Interfaces Without Perturbation
[Image: see text] To investigate the cyanylated cysteine vibrational probe group’s ability to report on binding-induced changes along a protein–protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light cha...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2018
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034165/ https://www.ncbi.nlm.nih.gov/pubmed/29787228 http://dx.doi.org/10.1021/acs.biochem.8b00283 |
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author | Dalton, Shannon R. Vienneau, Alice R. Burstein, Shana R. Xu, Rosalind J. Linse, Sara Londergan, Casey H. |
author_facet | Dalton, Shannon R. Vienneau, Alice R. Burstein, Shana R. Xu, Rosalind J. Linse, Sara Londergan, Casey H. |
author_sort | Dalton, Shannon R. |
collection | PubMed |
description | [Image: see text] To investigate the cyanylated cysteine vibrational probe group’s ability to report on binding-induced changes along a protein–protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques. |
format | Online Article Text |
id | pubmed-6034165 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-60341652018-07-08 Cyanylated Cysteine Reports Site-Specific Changes at Protein–Protein-Binding Interfaces Without Perturbation Dalton, Shannon R. Vienneau, Alice R. Burstein, Shana R. Xu, Rosalind J. Linse, Sara Londergan, Casey H. Biochemistry [Image: see text] To investigate the cyanylated cysteine vibrational probe group’s ability to report on binding-induced changes along a protein–protein interface, the probe group was incorporated at several sites in a peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques. American Chemical Society 2018-05-22 2018-07-03 /pmc/articles/PMC6034165/ /pubmed/29787228 http://dx.doi.org/10.1021/acs.biochem.8b00283 Text en Copyright © 2018 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Dalton, Shannon R. Vienneau, Alice R. Burstein, Shana R. Xu, Rosalind J. Linse, Sara Londergan, Casey H. Cyanylated Cysteine Reports Site-Specific Changes at Protein–Protein-Binding Interfaces Without Perturbation |
title | Cyanylated Cysteine Reports Site-Specific Changes
at Protein–Protein-Binding Interfaces Without Perturbation |
title_full | Cyanylated Cysteine Reports Site-Specific Changes
at Protein–Protein-Binding Interfaces Without Perturbation |
title_fullStr | Cyanylated Cysteine Reports Site-Specific Changes
at Protein–Protein-Binding Interfaces Without Perturbation |
title_full_unstemmed | Cyanylated Cysteine Reports Site-Specific Changes
at Protein–Protein-Binding Interfaces Without Perturbation |
title_short | Cyanylated Cysteine Reports Site-Specific Changes
at Protein–Protein-Binding Interfaces Without Perturbation |
title_sort | cyanylated cysteine reports site-specific changes
at protein–protein-binding interfaces without perturbation |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034165/ https://www.ncbi.nlm.nih.gov/pubmed/29787228 http://dx.doi.org/10.1021/acs.biochem.8b00283 |
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