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Published: Jun 20, 2018 DOI: 10.21769/BioProtoc.2878 Views: 6702
Edited by: Dennis Nürnberg Reviewed by: Sabu AbdulhameedPooja Saxena
Abstract
Small heat shock proteins (sHSP) are stress proteins which are ubiquitously found in almost all living organisms. They function as molecular chaperones, which assist in protein folding during translation and in the prevention of irreversible protein aggregation under denaturing conditions. This protocol describes the use of α-amylase as target protein in assessing the chaperone activity of wild and mutant recombinant small heat shock proteins of Mycobacterium leprae. Chaperone activity of these proteins, along with α-crystallin, a standard sHSP was demonstrated using a new method employing their protective effect against heat denaturation of α-amylase from porcine pancreas. The regained enzymatic activity of the α-amylase was demonstrated on starch agar plates stained with Iodine-Potassium Iodide (I2-KI) solution.
Keywords: Small heat shock proteinsBackground
Heat shock proteins (HSPs) are a conserved group of proteins which are induced when cells are exposed to external stress including heat and cold stress. Most of the members in this group are functionally related and are involved in the protein folding and unfolding mechanism. Small heat shock proteins (sHSPs) are a subset of HSPs with a molecular size ranging from 12 to 43 kDa and a conserved C-terminal region, called ‘α-crystalline domain’. The sHSPs show ATP independent molecular chaperone activity by binding to partially unfolded proteins and preventing their complete denaturation. There are several methods for demonstrating in vitro chaperone activity of sHSPs, using various substrate proteins like RuBisCO (Goloubinoff et al., 1989), rhodanese (Mendoza et al., 1992), insulin (Farahbakhsh et al., 1995), lysozyme (Rozema and Gellman, 1996), malate dehydrogenase (Lee et al., 1997), citrate synthase (Grallert et al., 1998), xylose reductase (Rawat and Rao, 1998), sorbitol dehydrogenase (Marini et al., 2000) and luciferase (Bepperling et al., 2012) etc. In these assays, the protective activity of sHSPs or other molecular chaperones is demonstrated based on their efficiency in refolding and prevention of aggregation during heat or chemical denaturation. These substrates have different quaternary structures, different rates of folding, and different tendencies to undergo irreversible side reactions during denaturation and HSP assisted renaturation. It has also been shown that protection against heat-inactivated restriction enzymes like NdeI and SmaI can be used to demonstrate the chaperone activity of α-crystallin in vitro (Hess and FitzGerald, 1998; Santhoshkumar and Sharma, 2001). HSP18 is one of the major immunodominant antigens of Mycobacterium leprae, and has been functionally characterized as an sHSP (Lini et al., 2008). In this protocol, we describe a simple method to assay chaperone activity of small heat shock proteins using heat inactivated α-amylase. The efficiency of sHSPs in preventing heat denaturation of α-amylase is demonstrated on starch agar plates after staining with an I2-KI solution. The iodine reagent binds to the starch polymer to form a dark blue color in the starch agar plates. Clear halos surrounding each well with sample are indicative of active amylase enzyme, which digests the starch in the agar plate (Atlas et al., 1995). The advantage of this method is its simplicity and the empirical demonstration of results on starch agar plates.
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Category
Biochemistry > Protein > Activity
Microbiology > Microbial biochemistry > Protein
Molecular Biology > Protein > Protein-protein interaction
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