title: Rapid collapse into a molten globule is followed by simple two-state kinetics in the folding of lysozyme from bacteriophage λ.
abstract: Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional NMR and electrospray ionization mass spectrometry, to investigate the folding kinetics of lysozyme from bacteriophage λ (λ lysozyme) at pH 5.6, 20 °C. The first step in the folding of λ lysozyme occurs very rapidly (τ < 1 ms) after refolding is initiated and involves both hydrophobic collapse and formation of a high content of secondary structure but only weak protection from (1)H/(2)H exchange and no fixed tertiary structure organization. This early folding step is reflected in the dead-time events observed in the far-UV CD and ANS fluorescence experiments. Following accumulation of this kinetic molten globule species, the secondary structural elements are stabilized and the majority (ca. 88%) of refolding molecules acquire native-like properties in a highly cooperative two-state process, with τ = 0.15 ± 0.03 s. This is accompanied by the acquisition of substantial native-like protection from hydrogen exchange. A double-mixing experiment and the absence of a denaturant effect reveal that slow (τ = 5 ± 1 s) folding of the remaining (ca. 12%) molecules is rate limited by the cis/trans isomerization of prolines that are trans in the folded enzyme. In addition, native state hydrogen exchange and classical denaturant unfolding experiments have been used to characterize the thermodynamic properties of the enzyme. In good agreement with previous crystallographic evidence, our results show that λ lysozyme is a highly dynamic protein, with relatively low conformational stability (ΔG°(N-U) = 25 ± 2 kJ·mol(-1)).
authors.affiliation: Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie B6, 4000 Liège (Sart Tilman), Belgium.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Di Paolo
authors.forename: Dorothée
authors.initials: D
authors.lastname: Balbeur
authors.forename: Edwin
authors.initials: E
authors.lastname: De Pauw
authors.forename: Christina
authors.initials: C
authors.lastname: Redfield
authors.forename: André
authors.initials: A
authors.lastname: Matagne
mesh_terms: D010582:Bacteriophage lambda / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D009113:Muramidase