title: Identification of multiple folding pathways of monellin using pulsed thiol labeling and mass spectrometry.
abstract: Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
authors.forename: Santosh Kumar
authors.initials: SK
authors.lastname: Jha
authors.forename: Amrita
authors.initials: A
authors.lastname: Dasgupta
authors.forename: Pooja
authors.initials: P
authors.lastname: Malhotra
authors.forename: Jayant B
authors.initials: JB
authors.lastname: Udgaonkar
mesh_terms: D003545:Cysteine / Q000737:chemistry; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry*; D017510:Protein Folding; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013194:Staining and Labeling; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014364:Tryptophan
chemical_list: D010940:Plant Proteins; D012997:Solvents; D013438:Sulfhydryl Compounds; C014674:methyl methanethiosulfonate; D014364:Tryptophan; D008741:Methyl Methanesulfonate; D003545:Cysteine