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Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli
Published on: December 26, 2020
Lysine acetylation of Hsp16.3: Effect on its structure, chaperone function and influence towards the growth of
Subhashree Barik1, Alok Kumar Panda2, Viplov Kumar Biswas3
1School of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar, India.
Abstract:
Hsp16.3 plays a vital role in the slow growth of Mycobacterium tuberculosis via its chaperone function. Many secretory proteins, including Hsp16.3 undergo acetylation in vivo. Seven lysine (K) residues (K64, K78, K85, K114, K119, K132 and K136) in Hsp16.3 are acetylated inside pathogen. However, how lysine acetylation affects its structure, chaperone function and pathogen's growth is still elusive. We examined these aspects by executing in vitro chemical acetylation (acetic anhydride modification) and by utilizing a lysine acetylation mimic mutant (Hsp16.3-K64Q/K78Q/K85Q/K114Q/K119Q/K132Q/K136Q). Far- and near-UV CD measurements revealed that the chemically acetylated proteins(s) and acetylation mimic mutant has altered secondary and tertiary structure than unacetylated/wild-type protein. The chemical modification and acetylation mimic mutation also disrupted the oligomeric assembly, increased surface hydrophobicity and reduced stability of Hsp16.3, as revealed by GF-HPLC, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding and urea denaturation experiments, respectively. These structural changes collectively led to an enhancement in chaperone function (aggregation and thermal inactivation prevention ability) of Hsp16.3. Moreover, when the H37Rv strain expressed the acetylation mimic mutant protein, its growth was slower in comparison to the strain expressing the wild-type/unacetylated Hsp16.3. Altogether, these findings indicated that lysine acetylation improves the chaperone function of Hsp16.3 which may influence pathogen's growth in host environment.
Insights
Lysine acetylation enhances the chaperone function of heat shock protein 16.3 (Hsp16.3) in Mycobacterium tuberculosis. This modification improves protein stability and slows pathogen growth, offering insights into tuberculosis pathogenesis.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Heat shock protein 16.3 (Hsp16.3) is crucial for the slow growth of Mycobacterium tuberculosis.
- Acetylation of lysine residues is a common post-translational modification in secretory proteins, including Hsp16.3.
- The impact of lysine acetylation on Hsp16.3's structure, function, and role in pathogen growth remains largely unknown.
Purpose of the Study:
- To investigate the effects of lysine acetylation on Hsp16.3 structure and chaperone activity.
- To determine how these modifications influence the growth of Mycobacterium tuberculosis.
Main Methods:
- In vitro chemical acetylation of Hsp16.3 using acetic anhydride.
- Construction and analysis of an Hsp16.3 acetylation mimic mutant (Hsp16.3-K64Q/K78Q/K85Q/K114Q/K119Q/K132Q/K136Q).
- Spectroscopic techniques (CD), chromatography (GF-HPLC), binding assays, and urea denaturation were employed to assess structural and functional changes.
Main Results:
- Chemical acetylation and the mimic mutation altered Hsp16.3's secondary and tertiary structures.
- Acetylation disrupted oligomeric assembly, increased surface hydrophobicity, and reduced protein stability.
- Enhanced chaperone activity was observed, with improved ability to prevent protein aggregation and thermal inactivation.
- Expression of the acetylation mimic mutant in Mycobacterium tuberculosis H37Rv led to slower pathogen growth.
Conclusions:
- Lysine acetylation significantly enhances the chaperone function of Hsp16.3.
- These acetylation-induced functional improvements may contribute to the slow growth of Mycobacterium tuberculosis within the host environment.
- Understanding Hsp16.3 acetylation provides potential avenues for therapeutic strategies against tuberculosis.
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