Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants

Abstract

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.

Fibrinogen stability under surfactant interaction

“…As a rule, most proteins follow the same sequence of binding events: initial interaction between surfactants [13] and proteins, partial unfold, exposing of more binding sites, and subsequent expansion of the polypeptide chain. However, the exact number of binding events and associated conformational changes will depend on the structure and amino acid composition of the protein in question [14][15][16].…”

Inactivation and Unfolding of the Hyperthermophilic Inorganic Pyrophosphatase from Thermus thermophilus by Sodium Dodecyl Sulfate

“…In this case, SDS can bind to the protein at a high molecule ratio (about one SDS molecule to two amino acid residues) mainly driven by hydrophobic interactions [8]. The behavior of proteins in solutions with moderate concentrations of SDS has been proposed to involve specific binding [9–14] and to behave quite differently [17]. The results herein also suggested that SDS could specifically bind to T-PPase and act as an inhibitor.…”

“…For a fully-denatured protein, SDS can cooperatively bind to the protein at a high molecule ratio [8]. SDS can also specifically or non-cooperatively bind to some proteins [9–14] and in some cases, activate enzymes [15,16]. Recently, it was found that proteins behaved quite differently when denatured by moderate concentrations of SDS [17].…”

The diversity of FtsY‐lipid interactions

“…FtsY was incubated for 5 or 60 min with lipid before trypsin digestion to examine the binding kinetics of interaction between FtsY and lipid. N‐terminal sequencing was performed as described,19 with the only difference that we used CAPS buffer (10 m M CAPS, pH 11) instead of Towbin buffer (25 m M Tris,192 m M glycine, 20% methanol, pH 8.3) containing 0.01% SDS to transfer from SDS–PAGE gel to PVDF membrane.…”