Investigating protein unfolding kinetics by pulse proteolysis

Abstract

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed ''pulse proteolysis'' to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP maltose complex, we have determined the dissociation equilibrium constant (K d ) of the complex from unfolding kinetic constants, which is also in good agreement with known K d values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.

Quantitative determination of ribosome nascent chain stability

“…Although we believe this method to be widely applicable, when applying it to new systems, it is important to take into account the limitations of pulse proteolysis, including possible steric hindrance because of biomolecular interactions (16,32). Nevertheless, it should be possible to extend our approach to determine how the ribosome modulates protein folding kinetics by monitoring unfolding rates via pulse proteolysis (33). In addition, given the seemingly important role of quality control at the ribosome in general cellular proteostasis (34), it will be important to know how RNC stability is altered in the presence of ribosome-associated chaperones such as trigger factor (in bacteria) or the ribosome quality control complex (in eukaryotes).…”

Age-Related Macular Degeneration-Associated Silent Polymorphisms in HtrA1 Impair Its Ability To Antagonize Insulin-Like Growth Factor 1

“…These precedent studies suggested that dSNP HtrA1, which is translated from a transcript containing NvAMD-associated SNPs, may also have an altered protein conformation. To consider this possibility, we subjected WT and dSNP HtrA1 produced in HEK 293T cells to partial proteolysis (8,(48)(49)(50). dSNP HtrA1 was more susceptible to proteolysis than WT HtrA1 was (Fig.…”

An Intramolecular Chaperone Inserted in Bacteriophage P22 Coat Protein Mediates Its Chaperonin-independent Folding

“…been used previously to determine protein stability (38) and monitor unfolding kinetics (39). Here we show that this technique can also be modified to monitor refolding kinetics and in a double jump experiment.…”

“…The refolding jump is then initiated by dilution into buffer. Thermolysin pulse proteolysis, which exploits the change in proteolytic susceptibility between folded and unfolded proteins (38,39), was used to measure the amount of native I-domain at varying times after refolding was initiated (Fig. 7b).…”