Abstract
The topologically similar βγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in βγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of βγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across βγ-crystallin members critical for their specialized functions.
Highlights
There has been a great deal of interest in understanding the evolution of diversity in protein domains, because similar folds appear to have a common ancestral origin
It would be interesting to understand how a noncanonical vertebrate motif diverged from a canonical Ca2ϩbinding motif of microbial ␥-crystallins during evolution attaining elevated domain stability
Evolution of Stability in ␥-Crystallin Superfamily—The vertebrate lens ␥-crystallins are evolved as innately stable proteins [3]
Summary
␥-Crystallin Domains—The regions of interest (21–108 amino acids) of the protein YP_002298252 (annotated as a hypothetical protein) from Rhodospirillum centenum (obtained from DSMZ, Germany) and Vibrillin (residue 174 –256 from a hypothetical protein with accession number NP_230470) from Vibrio cholerae were amplified and cloned in pET-21a expression vector (Novagen). Vibrillin and Flavollin mutants) and 0 M urea and eluted using a linear gradient of 0 –1.5 M NaCl. The Clostrillin and its mutants were purified from the soluble fraction on a Q-Sepharose column using 50 mM Tris-HCl, pH 9.9, 100 mM KCl. Equilibrium Unfolding—Equilibrium unfolding was studied for 26 proteins using GdmCl in the range of 0 – 6 M concentration with a step of 0.1 M (a total of 70 data points for each unfolding transition) as required for best fit [24] in the apo (Ca2ϩ-free, with 1 mM EDTA) and holo (with 5 mM CaCl2) in 50 mM Tris buffer, pH 7.0, containing 100 mM KCl. Unfolding was monitored by following the changes in the intrinsic Trp emission fluorescence by exciting at 295 nm. Where R is the universal gas constant; T is the absolute temperature; [D] is the molar concentration of denaturant; ⌬G0u is the free energy of unfolding; m is the dependence of ⌬G0u on denaturant concentration; Yobs is observed structural signal change; YN and Yu are the spectroscopic signals at the native and denatured state, respectively
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