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

Ben Moree, Katelyn Connell, Richard B. Mortensen, C. Tony Liu, Stephen Benkovic, Joshua Salafsky

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)806-815
Number of pages10
JournalBiophysical journal
Volume109
Issue number4
DOIs
StatePublished - Sep 10 2015

All Science Journal Classification (ASJC) codes

  • Biophysics

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    Moree, B., Connell, K., Mortensen, R. B., Liu, C. T., Benkovic, S., & Salafsky, J. (2015). Protein Conformational Changes Are Detected and Resolved Site Specifically by Second-Harmonic Generation. Biophysical journal, 109(4), 806-815. https://doi.org/10.1016/j.bpj.2015.07.016