Probing Lysozyme Conformation with Light Reveals a New Folding Intermediate

• Published in: Biochemistry (Easton) Vol. 44; no. 46; pp. 15139 - 15149
• Main Authors: Hamill, Andrea C, Wang, Shao-Chun, Lee, C. Ted
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22-11-2005
• Subjects: Azo Compounds - chemistry; Azo Compounds - radiation effects; Light; Muramidase - chemistry; Muramidase - radiation effects; Oxazines - chemistry; Oxazines - radiation effects; Photochemistry; Photosensitizing Agents - pharmacology; Protein Conformation - radiation effects; Protein Folding; Quaternary Ammonium Compounds - chemistry; Quaternary Ammonium Compounds - radiation effects; Scattering, Radiation; Spectrometry, Fluorescence; Spectrophotometry; Spectroscopy, Fourier Transform Infrared; Surface-Active Agents - chemistry; Surface-Active Agents - radiation effects
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi051646c

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 α domain of lysozyme, while the β domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known α-domain intermediate of lysozyme that contains a folded α domain and unfolded β 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.