Functional characterization of a small heat shock protein from Mycobacterium leprae

Nirmala, Lini; Rehna, Elengikal Abdul Azeez; Sugathan, Shiburaj; Jayapal, Jeya Maheshwari; Shankernarayan, Nallakandy Panagadan; Dharmalingam, Kuppamuthu

doi:10.1186/1471-2180-8-208

Cited by 21 publications

(29 citation statements)

References 39 publications

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“…The trypsin to chaperone ratio was 1 : 50 (w/w). molecular weight of HSP18P 52 earlier [28], in the present study, we only determined the molecular weight of HSP18S 52 by ESI-MS. All of the biophysical assays were performed with at least three independent protein preparations.…”

Section: Cloning Expression and Purification Of Two Hsp18 Variantsmentioning

confidence: 99%

“…We cloned and expressed wild-type and mutant HSP18 (HSP18S 52 and HSP18P 52 ) in E. coli M15 cells as described previously [27,28]. Wild-type and mutant cDNA sequences were confirmed by restriction digestion and DNA sequence analysis.…”

Section: Cloning Expression and Purification Of Two Hsp18 Variantsmentioning

confidence: 99%

“…Serine at position 52 mutates to proline. Subsequently, we characterized the HSP18 variant bearing proline at position 52 (HSP18P 52 ) [28]. Nine subunits of HSP18P 52 usually combine together to form a large oligomer of 173 kDa.…”

Section: Introductionmentioning

confidence: 99%

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A S52P mutation in the ‘α‐crystallin domain’ of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function

et al. 2013

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Mycobacterium leprae HSP18 is a small heat shock protein (sHSP). It is a major immunodominant antigen of M. leprae pathogen. Previously, we have reported the existence of two M. leprae HSP18 variants in various leprotic patients. One of the variants has serine at position 52, whereas the other one has proline at the same position. We have also reported that HSP18 having proline at position 52 ( HSP18P 52 ) is a nonameric protein and exhibits chaperone function. However, the structural and functional characterization of wild-type HSP18 having serine at position 52 ( HSP18S 52 ) is yet to be explored. Furthermore, the implications of the S52P mutation on the structure and chaperone function of HSP18 are not well understood. Therefore, we cloned and purified these two HSP18 variants. We found that HSP18S 52 is also a molecular chaperone and an oligomeric protein. Intrinsic tryptophan fluorescence and far-UV CD measurements revealed that the S52P mutation altered the tertiary and secondary structure of HSP18. This point mutation also reduced the oligomeric assembly and decreased the surface hydrophobicity of HSP18, as revealed by HPLC and 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid binding studies, respectively. Mutant protein was less stable against thermal and chemical denaturation and was more susceptible towards tryptic cleavage than wild-type HSP18. HSP18P 52 had lower chaperone function and was less effective in protecting thermal killing of Escherichia coli than HSP18S 52. Taken together, our data suggest that serine 52 is important for the larger oligomerization and chaperone function of HSP18. Because both variants differ in stability and function, they may have different roles in the survival of M. leprae in infected hosts. Abbreviations ACD, a-crystallin domain; bis-ANS, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt; CFU, colony-forming unit; FRET, F€ orster resonance energy transfer; Gu-HCl, guanidine hydrochloride; IPTG, isopropyl thio-b-D-galactoside; MDH, malate dehydrogenase; sHSP, small heat shock protein. Structured digital abstract

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Section: Cloning Expression and Purification Of Two Hsp18 Variantsmentioning

confidence: 99%

Section: Cloning Expression and Purification Of Two Hsp18 Variantsmentioning

confidence: 99%

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A S52P mutation in the ‘α‐crystallin domain’ of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function

et al. 2013

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“…The chaperone activity of the rice sHSPs was assayed using the restriction enzyme NdeI as a substrate (Lini et al 2008). Reaction mixtures consisting of 2 units of NdeI (New England Biolabs), its commercially available buffer and 1 μg sHSP were incubated for 90 min at temperatures ranging from 37 to 55°C.…”

Section: Biophysical Characterization Of Shspsmentioning

confidence: 99%

Characterization of rice small heat shock proteins targeted to different cellular organelles

Mani

Ramakrishna

Suguna

2015

Cell Stress and Chaperones

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Small heat shock proteins (sHSPs) are a family of ATP-independent molecular chaperones which prevent cellular protein aggregation by binding to misfolded proteins. sHSPs form large oligomers that undergo drastic rearrangement/dissociation in order to execute their chaperone activity in protecting substrates from stress. Substrate-binding sites on sHSPs have been predominantly mapped on their intrinsically disordered N-terminal arms. This region is highly variable in sequence and length across species, and has been implicated in both oligomer formation and in mediating chaperone activity. Here, we present our results on the functional and structural characterization of five sHSPs in rice, each differing in their subcellular localisation, viz., cytoplasm, nucleus, chloroplast, mitochondria and peroxisome. We performed activity assays and dynamic light scattering studies to highlight differences in the chaperone activity and quaternary assembly of sHSPs targeted to various organelles. By cloning constructs that differ in the length and sequence of the tag in the N-terminal region, we have probed the sensitivity of sHSP oligomer assembly and chaperone activity to the length and amino acid composition of the N-terminus. In particular, we have shown that the incorporation of an N-terminal tag has significant consequences on sHSP quaternary structure.

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“…The identification of these markers can contribute to the clinical diagnosis of TB and may also provide additional insight into the pathogenesis of TB (Kashyap et al, 2010). sHsp18 has been shown to be a major immunodominant antigen of M. leprae (Lini et al, 2008). Hsp65 has been shown to be an attractive marker for TB (Bothamley et al, 1992;Haldar et al, 2010;Lee et al, 1994;Rambukkana et al, 1991).…”

Section: Hsps In Tb Diagnosismentioning

confidence: 99%

Heat Shock Proteins in Mycobacterium tuberculosis: Involvement in Survival and Virulence of the Pathogen

Bajaj¹,

Batra²

2012

Understanding Tuberculosis - Deciphering the Secret Life of the Bacilli

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Functional characterization of a small heat shock protein from Mycobacterium leprae

Cited by 21 publications

References 39 publications

A S52P mutation in the ‘α‐crystallin domain’ of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function

A S52P mutation in the ‘α‐crystallin domain’ of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function

Characterization of rice small heat shock proteins targeted to different cellular organelles

Heat Shock Proteins in Mycobacterium tuberculosis: Involvement in Survival and Virulence of the Pathogen

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