Conserved folding pathways of α-lactalbumin and lysozyme revealed by kinetic CD, fluorescence, NMR, and interrupted refolding experiments

In this report, it is shown by a combination of stopped-flow CD, fluorescence, and time-resolved NMR studies that the Ca2+-induced refolding of bovine a-lactalbumin (BLA) at constant denaturant concentration (4 M urea) exhibits triple-exponential kinetics. In order to distinguish between parallel folding pathways and a strictly sequential formation of the native state, interrupted refolding experiments were conducted. We show here that the Ca2+-induced refolding of BLA involves parallel pathways and the transient formation of a folding intermediate on the millisecond timescale. Our data furthermore suggest that the two structurally homologous proteins BLA and hen egg white lysozyme share a common folding mechanism. We provide evidence that the guiding role of long-range interactions in the unfolded state of lysozyme in mediating intersubdomain interactions during folding is replaced in the case of BLA by the Ca2+-binding site. Time-resolved NMR spectroscopy, in combination with fast ion release from caged compounds, enables the measurement of complex protein folding kinetics at protein concentrations as low as 100 mu M and the concomitant detection of conformational transitions with rate constants of up to 8 s(-1). (C) 2008 Elsevier Ltd. All rights reserved.