title: Conformational change in the chromatin remodelling protein MENT.
abstract: Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is 'fine-tuned' in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding 'M-loop' and D-helix of MENT, but is independent of the protease specificity determining 'reactive centre loop'. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
authors.forename: Poh Chee
authors.initials: PC
authors.lastname: Ong
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Golding
authors.forename: Mary C
authors.initials: MC
authors.lastname: Pearce
authors.forename: James A
authors.initials: JA
authors.lastname: Irving
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Grigoryev
authors.forename: Debbie
authors.initials: D
authors.lastname: Pike
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Langendorf
authors.forename: Tanya A
authors.initials: TA
authors.lastname: Bashtannyk-Puhalovich
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Bottomley
authors.forename: James C
authors.initials: JC
authors.lastname: Whisstock
authors.forename: Robert N
authors.initials: RN
authors.lastname: Pike
authors.forename: Sheena
authors.initials: S
authors.lastname: McGowan
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002645:Chickens; D042002:Chromatin Assembly and Disassembly; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry; D005453:Fluorescence; D016297:Mutagenesis, Site-Directed; D009707:Nucleosomes / Q000378:metabolism; D010649:Phenylalanine; D011487:Protein Conformation; D014364:Tryptophan
chemical_list: D030161:Avian Proteins; D002868:Chromosomal Proteins, Non-Histone; C081441:MENT protein, Gallus gallus; D009707:Nucleosomes; D010649:Phenylalanine; D014364:Tryptophan; D004247:DNA