title: The presence of non-native helical structure in the unfolding of a beta-sheet protein MPT63.
abstract: MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β-sandwich protein containing 11 β strands and a very short stretch of 310helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β-sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV-CD and FTIR spectra clearly suggest that the low-pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β-sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non-native helical propensity of the protein structure.
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India.
authors.forename: Amrita
authors.initials: A
authors.lastname: Kundu
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India.
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Kundu
authors.affiliation: Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India.
authors.forename: Krishnananda
authors.initials: K
authors.lastname: Chattopadhyay
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D001426:Bacterial Proteins; C103339:MPT63 antigen, Mycobacterium tuberculosis
keywords: fluorescence correlation spectroscopy; molecular dynamics simulation; protein aggregation; protein folding; single molecule fluorescence