The widespread use of triclosan in personal care products as an antimicrobial agent is leading to its alarming tissue-bioaccumulation including human brain. However, knowledge of its potential effects on the vertebrate nervous system is still limited. Here, we hypothesized that sublethal triclosan concentrations are potent enough to alter motor neuron structure and function in zebrafish embryos exposed for prolonged duration. In this study, zebrafish embryos were used as vertebrate-animal model. Prolonged exposure (up to 4 days) of 0.6 mg/L (LC50, 96 h) and 0.3 mg/L (
Understanding the structural basis of altered properties of proteins due to changes in temperature or pH provides useful insights in designing proteins with improved stability. Here we report the basis for the pH-dependent thermostability of the Bacillus subtilis lipase (Lip A) using spectroscopic and X-ray crystallographic studies. At pH values above 7, lipase denatures and aggregates when heated at temperatures above 45 degrees C. However, at pH below 6 lipase denatures upon heating but the activity and its native structure is completely recovered upon cooling. In order to obtain the structural basis of this unusual stability of lipase, we determined high-resolution crystal structures of the lipase in two different crystal forms at pH 4.5 and 5. These structures show linear oligomerization of lipase using only two types of dimeric associations and these inter-molecular interactions are completely absent in several crystal forms of wild-type and mutant proteins obtained at basic pH. In accordance with the crystallographic studies, spectroscopic investigations reveal an invariant secondary structure in the pH range of 4-10. Quaternary organization of lipase at low pH resulted in changes in the tryptophan environment and binding of 1-anilino-8-naphthalene sulfate (ANS) at low pH. Low pH stability of the lipase is not observed in the presence of sodium chloride (>0.2 M) indicating the importance of ionic interactions at low pH. Inter- and intra-molecular ionic interactions that occur at pH below 6.0 are proposed to trap the molecule in a conformation that allows its complete refolding upon cooling.
Title: Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase Acharya P, Rajakumara E, Sankaranarayanan R, Rao NM Ref: Journal of Molecular Biology, 341:1271, 2004 : PubMed
Variation in gene sequences generated by directed evolution approaches often does not assure a minimalist design for obtaining a desired property in proteins. While screening for enhanced thermostability, structural information was utilized in selecting mutations that are generated by error-prone PCR. By this approach we have increased the half-life of denaturation by 300-fold compared to the wild-type Bacillus subtilis lipase through three point mutations generated by only two cycles of error-prone PCR. At lower temperatures the activity parameters of the thermostable mutants are unaltered. High-resolution crystal structures of the mutants show subtle changes, which include stacking of tyrosine residues, peptide plane flipping and a better anchoring of the terminus, that challenge rational design and explain the structural basis for enhanced thermostability. The approach may offer an efficient and minimalist solution for the enhancement of a desired property of a protein.
Title: Crystallization and preliminary X-ray crystallographic investigations on several thermostable forms of a Bacillus subtilis lipase Rajakumara E, Acharya P, Ahmad S, Shanmugam VM, Rao NM, Sankaranarayanan R Ref: Acta Crystallographica D Biol Crystallogr, 60:160, 2004 : PubMed
Bacillus subtilis lipase loses activity above pH 10.5 and below pH 6.0. However, at low pH, i.e. below pH 5.0, the lipase acquires remarkable thermostability. Activity was unaltered for 2 h at 323 K at pH 4.0-5.0, although at pH values above 7.0 the activity was lost rapidly within minutes. Circular-dichroism studies indicate significant changes in the tertiary structure of the lipase, whereas the secondary-structural content remained unaltered. To elucidate the structural basis of the enhanced thermostability, three different forms have been crystallized at low pH along with three crystal forms of two thermostable mutants obtained using a directed-evolution approach.
