title: Mechanism of chaperone function in small heat-shock proteins. Fluorescence studies of the conformations of T4 lysozyme bound to alphaB-crystallin.
abstract: To further develop the mechanistic understanding of small heat-shock protein (sHSP) chaperone activity, we investigate the nature of the intermediate states recognized by alpha-crystallin and the conformations that are stably bound. The model substrates consist of a set of well characterized, destabilized T4 Lysozyme (T4L) mutants that have been shown to differentially bind alpha-crystallin in a manner that reflects their free-energy of unfolding. A new approach for the detection of complex formation is introduced based on the conformational sensitivity of the fluorescent probe bimane, site-specifically introduced in T4L. Emission spectra of bimane-labeled T4L reveal two distinct patterns of intensity changes upon binding that depend on the molar ratio of alpha-crystallin to T4L. This directly demonstrates the two-mode nature of the binding process by the alpha-crystallins. Biphasic binding isotherms, obtained and analyzed over a wide range of T4L concentrations, demonstrate a substantially quenched bimane fluorescence in the low affinity-bound T4L that is similar to the quenching level observed due to denaturant unfolding. Furthermore, the pattern of intensity changes that occur upon binding of a T4L variant, bimane-labeled at an alternative solvent-exposed site, establishes a direct correlation between the quenching level observed in binding and unfolding. The results can be interpreted in terms of a model where alpha-crystallin binds at least two conformationally distinct non-native states of T4L, one of which is substantially unfolded and is bound with low affinity. A high affinity binding mode to compact states may be relevant to chaperone function in the lens, where protein damage is unlikely to cause global unfolding.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Hasige A
authors.initials: HA
authors.lastname: Sathish
authors.forename: Richard A
authors.initials: RA
authors.lastname: Stein
authors.forename: Guangyong
authors.initials: G
authors.lastname: Yang
authors.forename: Hassane S
authors.initials: HS
authors.lastname: Mchaourab
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic; D005453:Fluorescence; D006360:Heat-Shock Proteins / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D018832:Molecular Chaperones / Q000502:physiology*; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D038203:alpha-Crystallin B Chain / Q000235:genetics / Q000378:metabolism*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D006360:Heat-Shock Proteins; D007202:Indicators and Reagents; D018832:Molecular Chaperones; D038203:alpha-Crystallin B Chain; C021149:bimanes; D009113:Muramidase