title: Analysis of the peanut agglutinin molten globule-like intermediate by limited proteolysis.
abstract: These studies attempt to characterize the molten globule-like intermediate in the unfolding pathway of peanut agglutinin (PNA). PNA is the only known example of a homotetrameric protein that lacks the 2,2,2 or the fourfold symmetry. Previous studies have shown that PNA describes a non two-state unfolding process populated with a clearly defined intermediate. The intermediate is monomeric and has lost most of its tertiary structure and has a substantial amount of secondary structure still intact, thus described as a molten-globule (MG)-like intermediate. It was also shown by isothermal titration calorimetry to bind to lactose and some other ligands with an affinity similar to that of the native protein. This paper describes limited protease cleavage experiments on the intermediate using trypsin and protease V8 for its structural characterization. There are two hydrophobic cores in the PNA subunit. These experiments suggest that in the MG-like intermediate, the second hydrophobic core, near the sugar-binding loop of the protein loosens up. This effect is significantly reduced by the presence of 90% saturating lactose, as deduced by a reduction in cleavage propensity. This is also supported by the gain in the tertiary structure as observed by near-UV CD.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, Karnataka, India.
authors.forename: Nivedita
authors.initials: N
authors.lastname: Mitra
authors.forename: Sharmistha
authors.initials: S
authors.lastname: Sinha
authors.forename: R Manjunatha
authors.initials: RM
authors.lastname: Kini
authors.forename: Avadhesha
authors.initials: A
authors.lastname: Surolia
mesh_terms: D000595:Amino Acid Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019887:Peanut Agglutinin / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000378:metabolism
chemical_list: D019887:Peanut Agglutinin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin