TY - JOUR
T1 - Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants
AU - Hansen, Jonas Høeg
AU - Petersen, Steen Vang
AU - Andersen, Kell Kleiner
AU - Enghild, Jan J
AU - Damhus, Ture
AU - Otzen, Daniel
PY - 2009
Y1 - 2009
N2 - Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant-robust broad-specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS-PAGE and N-terminal sequencing. We observe well-defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for alpha-lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates. (c) 2008 Wiley Periodicals, Inc. Biopolymers 91: 221-231, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected].
AB - Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant-robust broad-specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS-PAGE and N-terminal sequencing. We observe well-defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for alpha-lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates. (c) 2008 Wiley Periodicals, Inc. Biopolymers 91: 221-231, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected].
U2 - 10.1002/bip.21125
DO - 10.1002/bip.21125
M3 - Journal article
C2 - 19072990
SN - 0006-3525
VL - 91
SP - 221
EP - 231
JO - Biopolymers
JF - Biopolymers
IS - 3
ER -