Four-State Equilibrium Unfolding of an scFv Antibody Fragment

Abstract

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.

Buffer-Dependent Fragmentation of a Humanized Full-Length Monoclonal Antibody

“…In large, multi-domain proteins such as an IgG, domain-domain interactions make a significant contribution to the overall stability of the molecule. 36,37,38,39,40 …”

Evidence of non-functional redundancy between two pea h-type thioredoxins by specificity and stability studies

“…2 of Supplementary Material), we could use the same equation described above for each of the two transitions (Pedroso et al, 2002). However, to test this assumption, we also carried out a three-state analysis of the GdmCl-denaturation data.…”

Structure of Stable Protein Folding Intermediates by Equilibrium ϕ-Analysis: The Apoflavodoxin Thermal Intermediate

“…4,8,28,38,44 In one recent work, the unfolding equilibrium of a single chain Fv antibody fragment was described as four-state. 5 For any protein displaying non-functional equilibrium intermediates between the native and unfolded states the energy difference that confines the polypeptide conformation into the active conformation is the free energy difference between the native state and the first non-functional intermediate. This free energy difference has been termed the relevant stability of the protein, 45 as opposed to the residual stability of the intermediate (relative to the unfolded state).…”

“…non-physiological pH, pressure or temperature), in the presence of high concentrations of certain molecules (denaturants, salts), or as a consequence of mutations. [1][2][3][4][5][6][7][8][9] The occurrence of equilibrium intermediates is sometimes associated with wellcharacterized stress phenomena, where specific physiological responses tend to reduce their harmful effects [10][11][12] and, in some cases, they are linked to human diseases, such as spongiform encephalopathy and other types of amyloidosis. 13,14 For some proteins, however, physiological roles have been postulated for their intermediate conformations.…”