The thermal behavior of gamma II, gamma IIIA, gamma IIIB, and gamma IVA crystallin, from calorimetric and spectral studies, has been analyzed in terms of selective unfolding of domains, interdomain interactions, conformational stability, and the existence of intermediates in the order-disorder transition equilibrium. The major endothermic transition (Tm) observed calorimetrically for all four fractions occurs between 67 and 78 degrees C, with enthalpy change (delta H) from 80 to 150 kcal/mol, values that agree reasonably well with those from spectroscopic measurements. gamma II and gamma IIIB show a second thermal event at T less than Tm whereas gamma IIIA and gamma IVA showed no additional transition. Urea-induced equilibrium unfolding of gamma II at acidic pH, unlike gamma IVA, is biphasic as monitored by CD and fluorescence, indicating the existence of an intermediate. The absence of a cooperative transition in gamma IVA in acidic urea and the appearance of a single endotherm in differential scanning calorimetry at low pH have been attributed to a structured intermediate that melts at low temperature. The difference in the folding/unfolding of gamma II and gamma IVA has been explained by subtle differences in the packing arrangement of their two domains and interactions between them. Thermal aggregation of gamma-crystallins could be prevented either by preincubation with ionic detergents or at low pH or in the presence of chemical denaturant, indicating that the protein surface charge and solvent polarity influence their stability. An increase in the 8-anilino-1-naphthalenesulfonate-bound fluorescence during heat denaturation also suggests that the thermal aggregation is governed by hydrophobic interactions. Study holds ProTherm entries: 5147, 5148, 5149, 5150, 5151 Extra Details: transition 1
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:28 p.m.
|Number of data points||15|
|Proteins||Gamma-crystallin B ; Gamma-crystallin B|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence GKITFYEDRGFQGHCYECSSDCPNLQPYFSRCNSIRVDSGCWMLYERPNYQGHQYFLRRGDYPDYQQWMGFNDSIRSCRLIPQHTGTFRMRIYERDDFRGQMSEITDDCPSLQDRFHLTEVHSLNVLEGSWVLYEMPSYRGRQYLLRPGEYRRYLDWGAMNAKVGSLRRVMDFY|