Polypeptide Chain Unfolding
Polypeptide Chain Unfolding - Related Videos
09:42
Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
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Cited by 6 •
2012
We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.
10:15
Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
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Cited by 2 •
2012
A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.
01:37
The Unfolded Protein Response
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2023
The ER is the hub of protein synthesis in a cell. It has robust systems to quality control protein folding and also for degradation of terminally misfolded proteins. Under normal conditions, a small proportion of misfolded proteins that cannot be salvaged need to be transported to the cytoplasm by the ER-associated degradation or ERAD pathways. However, if the ERAD cannot handle the misfolded proteins, the cell activates the unfolded protein response or UPR to adjust the protein folding...
07:35
Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli
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Cited by 48 •
2014
Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.
02:38
Assessing Bacterial Chaperone Activity via Thermal Unfolding of a Model Protein
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2025
Source: Dahl, J. et al. Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo. J. Vis. Exp. (2016)This video demonstrates a fluorescence spectrophotometer-based assay to evaluate chaperone activity on a substrate protein under acid and heat stress. It outlines the steps for monitoring substrate unfolding and aggregation through light scattering, comparing conditions with and without the chaperone.
FAQs
Unfolding begins when stabilizing noncovalent interactions are disrupted. Hydrogen bonds help maintain secondary structure, while ionic interactions and hydrophobic packing contribute to higher-order organization. As these contacts weaken, the chain can adopt less organized conformations and previously buried residues may become exposed. This structural change explains why a protein may lose activity even though its amino acid sequence is unchanged.
These conditions perturb the forces that maintain a protein’s native arrangement, but they do so as environmental challenges rather than by altering the encoded amino acid sequence. The resulting loss of structural organization can expose residues that were normally hidden. Studying different conditions helps connect environmental stress with changes in protein activity, stability, and the tendency to aggregate.
Return to favorable conditions may allow a polypeptide chain to refold, but exposed residues can also promote interactions between chains. Those interactions may produce aggregates instead of restoring the original arrangement. This contrast makes refolding outcome an important indicator of protein stability and links molecular structure to broader questions about whether function can be recovered after stress.
A useful investigation compares a protein under favorable conditions with samples exposed to heat, extreme pH, or a chemical denaturant, then examines changes in structure, biological activity, exposure of normally buried residues, and aggregation. Returning conditions to a more favorable state can further reveal whether refolding occurs. Together, these observations distinguish structural disruption from recovery or persistent misfolding.
When proteins lose their native organization, cells must respond to altered activity and the possibility of aggregation. The study of unfolding therefore helps explain how protein quality control and cellular stress responses address destabilized proteins. It also provides context for misfolding-related disease, where abnormal structural states can have consequences beyond a single protein’s immediate loss of function.
Unfolding behavior reveals which conditions compromise protein stability and whether function can return after those conditions change. That information supports the design of more stable proteins for research and biotechnology. It also helps interpret why a protein may aggregate or lose activity during use, making structural stability a practical consideration when developing or applying protein-based systems.