Alternatively folded states of an immunoglobulin

Abstract

Well-defined, non-native protein structures of low stability have been increasingly observed as intermediates in protein folding or as equilibrium structures populated under specific solvent conditions. These intermediate structures, frequently referred to as molten globule states, are characterized by the presence of secondary structure, a lack of significant tertiary contacts, increased hydrophobicity and partial specific volume as compared to native structures, and low cooperativity in thermal unfolding. The present study demonstrates that under acidic conditions (pH less than 3) the antibody MAK33 can assume a folded stable conformation. This A-state is characterized by a high degree of secondary structure, increased hydrophobicity, a native-like maximum wavelength of fluorescence emission, and a tendency toward slow aggregation. A prominent feature of this low-pH conformation is the stability against denaturant and thermal unfolding that is manifested in highly cooperative reversible phase transitions indicative of the existence of well-defined tertiary contacts. These thermodynamic results are corroborated by the kinetics of folding from the completely unfolded chain to the alternatively folded state at pH 2. The given data suggest that MAK33 at pH 2 adopts a cooperative structure that differs from the native immunoglobulin fold at pH 7. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its extraordinary structural stability that is characteristic for native protein structures.

Thermodynamic and functional characterization of a stable IgG conformer obtained by renaturation from a partially structured low pH‐induced state

“…1); a similar deconvolution pattern comprising five subtransitions has been previously obtained for mouse monoclonal IgG1 [13]. Upon acidification of the IgG solution to pH 2, the protein retains a r-stranded secondary structure ( elements might have taken place.…”

“…Detailed analysis of pH-stability of rabbit IgG in the pH 7-2 range by DSC confirms the lack of well-defined structure of the CH2 domain at pH < 3 (Martsev, Kravchuk and Vlasov, manuscript in preparation). Recently, mouse monoclonal IgG1 antibody MAK33 has been shown to adopt, at pH 2, the so-called A-state which was characterized as an alternatively folded molten globule-like state [13]. Comparison of calorimetric data obtained in our own and previous studies [13] reveals that the A-state is clearly distinct from the partially structured (intermediate) state of rabbit IgG at pH 2 when considering overall AH and TM of thermal unfolding transition, and the number of subtransitions under the resulting melting curve.…”

“…Recently, mouse monoclonal IgG1 antibody MAK33 has been shown to adopt, at pH 2, the so-called A-state which was characterized as an alternatively folded molten globule-like state [13]. Comparison of calorimetric data obtained in our own and previous studies [13] reveals that the A-state is clearly distinct from the partially structured (intermediate) state of rabbit IgG at pH 2 when considering overall AH and TM of thermal unfolding transition, and the number of subtransitions under the resulting melting curve. Our calorimetric and functional studies indicate that reversibility of the low pH-induced transition of rabbit IgG from native (N) to intermediate (I) state is incomplete and renaturation yields a stable native-like N~ conformer of IgG according to the scheme: N ~ I--~ N I.…”

Domain interactions stabilize the alternatively folded state of an antibody Fab fragment

“…Recently, we investigated the structure of an intact antibody at low pH. Although some of the characteristics of a molten globule could be observed, the antibody exhibited a high stability against gdm/C1 and temperature, indicating a cooperative denaturation of a at least partially defined tertiary structure which we, therefore, termed alternatively folded state [11]. In this study we have extended the investigations to the Fab fragment of the respective antibody.…”

Characterization of Monomeric 4‐Aminobutyrate Aminotransferase at Low pH

“…Buchner et al [20] have shown that at pH values below 3.0, a murine monoclonal antibody (MAK 33) acquires a stable conformation that is different from both the native and denatured state. This alternative folded state exhibits certain characteristics of the molten globule state, but it differs from it by its remarkable stability.…”