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Issue 42, 2007
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Second-harmonic generation for studying structural motion of biological molecules in real time and space

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Abstract

SHG and sum-frequency generation (SFG) are surface-selective, nonlinear optical techniques whose ability to measure the average tilt angle of molecules on surfaces is well known in non-biological systems. By labeling molecules with a second-harmonic-active dye probe, SHG detection is extended to any biological molecule. The method has been used in previous work to detect biomolecules at an interface and their ligand-induced conformational changes. Here I demonstrate that SHG can be used to study structural motion quantitatively using a probe placed at a specific site (Cys-77) in adenylate kinase, a protein. The protein is also labeled non-site-specifically via amines. Labeled protein is absorbed to a surface and a baseline SH signal is measured. Upon introducing ATP, AMP or a specific inhibitor, AP5A, the baseline signal changes depending on the ligand and the labeling site. In particular, a substantial change in SH intensity is produced upon binding ATP to the amine-labeled protein, consistent with the X-ray crystal structures. In contrast, SHG polarization measurements are used to quantitatively determine that no rotation occurs at site Cys-77, in agreement with the lack of motion observed at this site in the X-ray crystal structures. A method for building a global map of conformational change in real time and space is proposed using a set of probes placed at different sites in a biomolecule. For this purpose, SH-active unnatural amino acids are attractive complements to exogenous labels.

Graphical abstract: Second-harmonic generation for studying structural motion of biological molecules in real time and space

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Article information


Submitted
10 Jul 2007
Accepted
29 Aug 2007
First published
07 Sep 2007

Phys. Chem. Chem. Phys., 2007,9, 5704-5711
Article type
Paper

Second-harmonic generation for studying structural motion of biological molecules in real time and space

J. S. Salafsky, Phys. Chem. Chem. Phys., 2007, 9, 5704
DOI: 10.1039/B710505C

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