Using a second‐order differential model to fit data without baselines in protein isothermal chemical denaturation

Abstract

In vitro protein stability studies are commonly conducted via thermal or chemical denaturation/renaturation of protein. Conventional data analyses on the protein unfolding/(re)folding require well-defined pre-and post-transition baselines to evaluate Gibbs free-energy change associated with the protein unfolding/(re)folding. This evaluation becomes problematic when there is insufficient data for determining the pre-or post-transition baselines. In this study, fitting on such partial data obtained in protein chemical denaturation is established by introducing second-order differential (SOD) analysis to overcome the limitations that the conventional fitting method has. By reducing numbers of the baseline-related fitting parameters, the SOD analysis can successfully fit incomplete chemical denaturation data sets with high agreement to the conventional evaluation on the equivalent completed data, where the conventional fitting fails in analyzing them. This SOD fitting for the abbreviated isothermal chemical denaturation further fulfills data analysis methods on the insufficient data sets conducted in the two prevalent protein stability studies.

The uniqueness of flow in probing the aggregation behavior of clinically relevant antibodies

“…We also showed that commonly employed in silico methods cannot act as a surrogate for experimental measurements of flow‐induced aggregation propensity. We have previously shown that extensional flow triggers partial unfolding of BSA, 59 which may expose APRs that are sequestered from solvent in the thermodynamically stable native protein (Δ G UN = 15.2 kJ mol −1 ), 82 thereby evading detection by both CamSol and Solubis. In this regard, it is interesting to note that many of the APRs predicted by Solubis 26 are found on the V H ‐ V L interface or the CDRH3 loop (Figure S6b).…”

Snapshots of urea‐induced early structural changes and unfolding of an ankyrin repeat protein at atomic resolution

“…Intrinsic tryptophan fluorescence of PSMD10 Gankyrin was collected using PR.ChemControl software of Nanotemper Technologies to monitor chemical denaturation. We calculated the slopes of the pre‐transition and post‐transition data points and used the equations as described earlier to determine fraction unfolded 40 . The data points were further fitted using four parametric equations in Graphpad V8.0.…”

“…We calculated the slopes of the pre-transition and post-transition data points and used the equations as described earlier to determine fraction unfolded. 40 The data points were further fitted using four parametric equations in Graphpad V8.0.…”

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases

“…The main controversial point of this method is the assumption that proteins are completely denatured under these experimental conditions, and such denaturation is completely reversible. However, a detailed analysis of the protocols of such experiments shows that most of the methods for assessing the unfolding of the protein structure are indirect and replete with numerous assumptions [ 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 ]. Thus, proteins that are supposed as fully unfolded are actually only partially unfolded, and the degree and nature of unfolding of the same protein can differ significantly depending on the denaturation protocol used [ 104 , 107 ].…”