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

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

doi:10.1016/j.bpj.2015.07.016

Joshua Salafsky, C Tony Liu + Show 4 more

Open Access

https://doi.org/10.1016/j.bpj.2015.07.016

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Journal: Biophysical Journal	Publication Date: Aug 1, 2015
Citations: 39	License type: cc-by

Affiliation: Pennsylvania State University

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.

Highlights

The relationship between protein structure and function has long been recognized as fundamental for understanding biological mechanisms
In the work presented here, we demonstrate a broadly applicable method based on Second-harmonic generation (SHG) for studying protein conformational changes upon ligand binding in real time and under physiological conditions
To explore the ability of our SHG bilayer system to detect and measure the conformational changes of a protein, we began by testing the system on CaM and maltose-binding protein (MBP), two proteins previously studied by SHG [34,35]

Summary

Introduction

The relationship between protein structure and function has long been recognized as fundamental for understanding biological mechanisms. Far from being static in structure, proteins are dynamic molecules that are capable of changing their shape, or conformation, in response to changes in their environment and upon ligand binding. Second-harmonic generation (SHG) is a nonlinear optical technique [5,6] in which two photons of equal energy are combined by a nonlinear material or molecule to generate one photon with twice the energy. This process is forbidden in a medium with centrosymmetry, but as symmetry is always broken at an interface, SHG is intrinsically surface selective. When second-harmonic-active molecules are immobilized at an interface and irradiated with a fundamental

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