Probing lysozyme conformation with light reveals a new folding intermediate.
A means to control lysozyme conformation with light illumination has been developed using the interaction of the protein with a photoresponsive surfactant. Upon exposure to the appropriate wavelength of light, the azobenzene surfactant undergoes a reversible photoisomerization, with the visible-light (trans) form being more hydrophobic than the UV-light (cis) form. As a result, surfactant binding to the protein and, thus, protein unfolding, can be tuned with light. Small-angle neutron scattering (SANS) measurements were used to provide detailed information of the protein conformation in solution. Shape-reconstruction methods applied to the SANS data indicate that under visible light the protein exhibits a native-like form at low surfactant concentrations, a partially swollen form at intermediate concentrations, and a swollen/unfolded form at higher surfactant concentrations. Furthermore, the SANS data combined with FT-IR spectroscopic analysis of the protein secondary structure reveal that unfolding occurs primarily in the alpha domain of lysozyme, while the beta domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known alpha-domain intermediate of lysozyme that contains a folded alpha domain and unfolded beta domain. Because the interactions between the photosurfactant and protein can be tuned with light, illumination with UV light returns the protein to a native-like conformation. Fluorescence emission data of the nonpolar probe Nile red indicate that hydrophobic domains become available for probe partitioning in surfactant-protein solutions under visible light, while the availability of these hydrophobic domains to the probe decrease under UV light. Dynamic light scattering and UV-vis spectroscopic measurements further confirm the shape-reconstruction findings and reveal three discrete conformations of lysozyme. The results clearly demonstrate that visible light causes a greater degree of lysozyme swelling than UV light, thus allowing for the protein conformation to be controlled with light.
Hamill
Andrea C
AC
Department of Chemical Engineering, University of Southern California, Los Angeles, California 90089-1211, USA.
Wang
Shao-Chun
SC
Lee
C Ted
CT
D001391:Azo Compounds / Q000737:chemistry / Q000528:radiation effects; D008027:Light; D009113:Muramidase / Q000737:chemistry* / Q000528:radiation effects*; D010078:Oxazines / Q000737:chemistry / Q000528:radiation effects; D010777:Photochemistry; D017319:Photosensitizing Agents / Q000494:pharmacology; D011487:Protein Conformation / Q000528:radiation effects; D017510:Protein Folding; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000528:radiation effects; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared; D013501:Surface-Active Agents / Q000737:chemistry / Q000528:radiation effects
D001391:Azo Compounds; D010078:Oxazines; D017319:Photosensitizing Agents; D000644:Quaternary Ammonium Compounds; D013501:Surface-Active Agents; D009113:Muramidase; C044808:nile red