Conserved folding pathways of alpha-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 Ca(2+)-induced refolding of bovine alpha-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 Ca(2+)-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 Ca(2+)-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 microM and the concomitant detection of conformational transitions with rate constants of up to 8 s(-1).
Schlepckow
Kai
K
Center for Biomolecular Magnetic Resonance, Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe University, Max-von-Laue-Str. 7, D-60438 Frankfurt/Main, Germany.
Wirmer
Julia
J
Bachmann
Annett
A
Kiefhaber
Thomas
T
Schwalbe
Harald
H
D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
D001051:Apoenzymes; D014508:Urea; D007768:Lactalbumin; C421864:hen egg lysozyme; D009113:Muramidase; D002118:Calcium