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Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation

We present here a straightforward, broadly applicable technique for real-time detection and measurement of protein conformational changes in solution. This method is based on tethering proteins labeled with a second-harmonic generation (SHG) active dye to supported lipid bilayers. We demonstrate our...

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Autores principales: Moree, Ben, Connell, Katelyn, Mortensen, Richard B., Liu, C. Tony, Benkovic, Stephen J., Salafsky, Joshua
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Biophysical Society 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547196/
https://www.ncbi.nlm.nih.gov/pubmed/26287632
http://dx.doi.org/10.1016/j.bpj.2015.07.016
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author Moree, Ben
Connell, Katelyn
Mortensen, Richard B.
Liu, C. Tony
Benkovic, Stephen J.
Salafsky, Joshua
author_facet Moree, Ben
Connell, Katelyn
Mortensen, Richard B.
Liu, C. Tony
Benkovic, Stephen J.
Salafsky, Joshua
author_sort Moree, Ben
collection PubMed
description We present here a straightforward, broadly applicable technique for real-time detection and measurement of protein conformational changes in solution. This method is based on tethering proteins labeled with a second-harmonic generation (SHG) active dye to supported lipid bilayers. We demonstrate our method by measuring the conformational changes that occur upon ligand binding with three well-characterized proteins labeled at lysine residues: calmodulin (CaM), maltose-binding protein (MBP), and dihydrofolate reductase (DHFR). We also create a single-site cysteine mutant of DHFR engineered within the Met20 catalytic loop region and study the protein’s structural motion at this site. Using published x-ray crystal structures, we show that the changes in the SHG signals upon ligand binding are the result of structural motions that occur at the labeled sites between the apo and ligand-bound forms of the proteins, which are easily distinguished from each other. In addition, we demonstrate that different magnitudes of the SHG signal changes are due to different and specific ligand-induced conformational changes. Taken together, these data illustrate the potential of the SHG approach for detecting and measuring protein conformational changes for a wide range of biological applications.
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spelling pubmed-45471962016-08-18 Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation Moree, Ben Connell, Katelyn Mortensen, Richard B. Liu, C. Tony Benkovic, Stephen J. Salafsky, Joshua Biophys J Proteins and Nucleic Acids We present here a straightforward, broadly applicable technique for real-time detection and measurement of protein conformational changes in solution. This method is based on tethering proteins labeled with a second-harmonic generation (SHG) active dye to supported lipid bilayers. We demonstrate our method by measuring the conformational changes that occur upon ligand binding with three well-characterized proteins labeled at lysine residues: calmodulin (CaM), maltose-binding protein (MBP), and dihydrofolate reductase (DHFR). We also create a single-site cysteine mutant of DHFR engineered within the Met20 catalytic loop region and study the protein’s structural motion at this site. Using published x-ray crystal structures, we show that the changes in the SHG signals upon ligand binding are the result of structural motions that occur at the labeled sites between the apo and ligand-bound forms of the proteins, which are easily distinguished from each other. In addition, we demonstrate that different magnitudes of the SHG signal changes are due to different and specific ligand-induced conformational changes. Taken together, these data illustrate the potential of the SHG approach for detecting and measuring protein conformational changes for a wide range of biological applications. The Biophysical Society 2015-08-18 2015-08-18 /pmc/articles/PMC4547196/ /pubmed/26287632 http://dx.doi.org/10.1016/j.bpj.2015.07.016 Text en © 2015 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Proteins and Nucleic Acids
Moree, Ben
Connell, Katelyn
Mortensen, Richard B.
Liu, C. Tony
Benkovic, Stephen J.
Salafsky, Joshua
Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation
title Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation
title_full Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation
title_fullStr Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation
title_full_unstemmed Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation
title_short Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation
title_sort protein conformational changes are detected and resolved site specifically by second-harmonic generation
topic Proteins and Nucleic Acids
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547196/
https://www.ncbi.nlm.nih.gov/pubmed/26287632
http://dx.doi.org/10.1016/j.bpj.2015.07.016
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