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Protein Folding01:25

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Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation, critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.
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The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
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ER is the primary site for the maturation and folding of soluble and transmembrane secretory proteins. The calnexin cycle is a specific chaperone system that folds and assesses the confirmation of N-glycosylated proteins before they can exit the ER lumen. The primary players of this quality check pipeline are the lectins, ER-resident chaperones, and a glucosyl transferase enzyme. In case the calnexin system in the lumen fails to salvage a misfolded protein, it is transported to the cytoplasm...
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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...
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Bacterial protein maturation is a tightly regulated process that ensures newly synthesized polypeptides achieve correct functional conformations. This maturation involves a series of modifications, folding events, and quality control steps, often assisted by specialized chaperone proteins.N-Terminal ModificationsThe maturation of bacterial polypeptides begins cotranslationally as the polypeptide exits the ribosome. The first amino acid, N-formylmethionine (fMet), is typically modified at the...
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Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
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Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
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Smart Protein Refolding System Based on UCST-Type Ureido Polymers.

Yamato Tanaka1, Chun Hao Niu1, Taira Sasaki1

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Ureido polymers with upper critical solution temperature (UCST) behavior prevent protein aggregation and promote refolding upon cooling. This cooling-induced liquid-liquid phase separation (LLPS) facilitates protein release and recovery with enzymatic activity.

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Area of Science:

  • Polymer Science
  • Biochemistry
  • Materials Science

Background:

  • Ureido polymers exhibit unique upper critical solution temperature (UCST)-type phase behavior, contrasting with typical lower critical solution temperature (LCST)-type polymers.
  • UCST-type polymers undergo liquid-liquid phase separation (LLPS) upon cooling, unlike the liquid-solid transition seen in LCST-type polymers.

Purpose of the Study:

  • To investigate the effect of cooling-induced LLPS of UCST-type ureido polymers on protein refolding.
  • To evaluate the potential of these polymers in preventing protein aggregation and facilitating refolding.

Main Methods:

  • Preparation of ureido polymers functionalized with hydrophobic undecyl groups.
  • Incubation of proteins with polymers at elevated temperatures, followed by cooling.
  • Analysis of protein aggregation, release, and enzymatic activity post-incubation.
  • Separation of refolded protein from precipitated polymers via centrifugation.

Main Results:

  • Ureido polymers functionalized with undecyl groups prevented protein aggregation during heating.
  • Cooling induced spontaneous protein release from the polymer, with the released protein retaining enzymatic activity.
  • Protein refolding efficiency was enhanced near the polymer's phase separation temperature.
  • Efficient separation of refolded protein from precipitated polymers was achieved through low-temperature centrifugation.

Conclusions:

  • UCST-type ureido polymers can be utilized for effective protein refolding and recovery.
  • The cooling-induced LLPS of these polymers plays a crucial role in facilitating protein release and refolding.
  • This approach offers a novel method for protein stabilization and purification.