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Analysis of βB1‐crystallin unfolding equilibrium by spin and fluorescence labeling: Evidence of a dimeric intermediate
Author(s) -
Koteiche Hanane A.,
Kumar M. Satish,
Mchaourab Hassane S.
Publication year - 2007
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2007.04.004
Subject(s) - crystallin , dimer , chemistry , fluorescence , folding (dsp implementation) , biophysics , population , protein folding , crystallography , equilibrium unfolding , protein subunit , native state , biology , biochemistry , physics , demography , organic chemistry , quantum mechanics , sociology , gene , electrical engineering , engineering
A central step in understanding lens aging is to characterize the thermodynamic stability of its proteins and determine the consequences of changes in the primary sequence on their folding equilibria. For this purpose, destabilized mutations were introduced in βB1‐crystallin targeting the domain interface within the fold of a subunit. Global unfolding was monitored by tryptophan fluorescence while concomitant structural changes at the dimer interface were monitored by fluorescence and spin labels. Both spectral probes report explicit evidence of multi‐state unfolding equilibrium. The biphasic nature of the unfolding curves was more pronounced at higher protein concentration. Distinct shifts in the midpoint of the second transition reflect the population of a dimeric intermediate. This intermediate may be a critical determinant for the life‐long stability of the β‐crystallins and has important consequences on interactions with α‐crystallin.