title: Unusual optical resolution of all four tryptophan residues in MPT63 protein by phosphorescence spectroscopy: assignment and significance.
abstract: MPT63, a secreted protein of unknown function that is specific to Mycobacterium tuberculosis and a potential drug target, contains four Tryptophan (Trp/W) residues located at positions 26, 48, 82, and 129 in the amino acid sequence. All of the four Trp residues have been optically resolved by simple inexpensive phosphorescence spectroscopy at 77 K. The protein architecture provides a delicate micro-environment and location of Trp residues giving rise to four different (0,0) bands in the phosphorescence spectra. Calculation of intra Trp energy transfer (ET) efficiency, accessible surface area (ASA) of Trp residues, and environment of Trp in the wild-type (WT) and the mutant W26F [where, Trp 26 is replaced by phenyl alanine (Phe/F)] reveal: E(T1) (W82) > E(T1) (W48) > E(T1) (W129) > E(T1) (W26), where E(T1) is the lowest (π-π*) triplet state energy of Trp. The (0,0) band observed at 421.6 nm assigned for Trp 26 is found to be the longest wavelength (0,0) band so far reported in the literature. Fluorescence in WT and W26F is dominated by buried or partially exposed Trp residues indicated by time-resolved spectra. Circular Dichroism (CD) studies and the time-resolved anisotropy measurement confirm the unaltered secondary and tertiary structure of the mutant compared to that of the WT. Excitation energy dependent phosphorescence spectra suggest that the intensity of the different (0,0) bands could be tuned and Tyrosine (Tyr/Y) residue is silent in emission. Optical resolution of all the Trp residues will help understand the role of each Trp residue in the folding/unfolding mechanism and in the interaction with other systems.
authors.affiliation: Structural Biology & Bio-Informatics Division, Indian Institute of Chemical Biology, Kolkata-700 032, India.
authors.forename: Ranendu
authors.initials: R
authors.lastname: Ghosh
authors.forename: Manini
authors.initials: M
authors.lastname: Mukherjee
authors.forename: Krishnananda
authors.initials: K
authors.lastname: Chattopadhyay
authors.forename: Sanjib
authors.initials: S
authors.lastname: Ghosh
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements; D008958:Models, Molecular; D009154:Mutation; D055620:Optical Phenomena; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; C103339:MPT63 antigen, Mycobacterium tuberculosis; D014364:Tryptophan