Second-harmonic generation for studying structural motion of biological molecules in real time and space.
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, AP(5)A, 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.
Salafsky
Joshua S
JS
Biodesy, LLC, Burlingame, CA 94010, USA.
D000249:Adenosine Monophosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000263:Adenylate Kinase / Q000737:chemistry; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D015226:Dinucleoside Phosphates / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation
D015226:Dinucleoside Phosphates; D000249:Adenosine Monophosphate; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase; D003545:Cysteine