[en] protein folding ; lysozyme ; hydrogen exchange ; NMR ; mass spectrometry ; circular dichroism
[en] 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 lambda (lambda lysozyme) at pH 5.6, 20 degrees C. The first step in the folding of lambda lysozyme occurs very rapidly (tau < 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 tau = 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 (tau = 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 lambda lysozyme is a highly dynamic protein, with relatively low conformational stability (DeltaG degrees (N-U) = 25 +/- 2 kJ.mol(-1)).