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The ubiquitin E2 variant domain of TSG101 (TSG101‐UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101‐UEV/PTAP interactions has emerged as a promising strategy for the development of host‐oriented broad‐spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101‐UEV/PTAP binding interface by searching for drug‐like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established by combining in vitro miniaturized HTS assays and a set of cell‐based activity assays including high‐content bimolecular complementation, virus‐like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101‐UEV/PTAP binding with IC50s in the low μM range and are able to disrupt the interaction between full‐length TSG101 and viral proteins in human cells and inhibit viral replication. State‐of‐the‐art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101‐UEV binding pockets. These inhibitors represent promising hits for the development of novel broad‐spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions. [ABSTRACT FROM AUTHOR]

In 1972 Christian B. Anfinsen received the Nobel Prize in Chemistry for "…his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation." The understanding of this principle is crucial for physical biochemistry students, since protein folding studies, bio-computing sciences and protein design approaches are founded on such a well-demonstrated connection. Herein, we describe a detailed and easy-to-follow experiment to reproduce the most relevant assays carried out at Anfinsen's laboratory in the 60s. This experiment provides students with a platform to interpret by themselves the structural and kinetic experiments conceived to understand the protein folding problem. In addition, this three-day experiment brings students a nice opportunity for protein manipulation as well as for the setting up of spectroscopic and chromatographic techniques.

A nascent simple polypeptide chain in vitro, 64-residue chymotrypsin inhibitor 2, does not display a tendency to form stable structure until it is synthesized into a complex form and the native secondary structural elements of alpha-helix and beta-sheet are absent in the structures formed in a nascent simple protein. Thus the structural growth in a simple protein is cooperative when compared to a hierarchical formation in a complex protein.

Experimental studies of two fragments of the barley chymotrypsin inhibitor 2 reveal a similarity in the transition states for protein folding and for the association between the two fragments. The mutant fragments obtained from the breaking up of mutant proteins aid these studies. The presence of hydrophobic residue leads to a lowering of the equilibrium constants for protein folding and fragment association. Further kinetic studies indicate that the rate constants for both these processes are almost the same, suggesting that the association between two protein fragments serves as a pathway for the study of protein folding.

A study of the early events in protein folding by the dissection of proteins into complementary fragments reveals the formation of a native-like structure from two fragments of chymotrypsin inhibitor 2 and CI2-(60-83), in a second-order reaction that combines collision and rearrangement. Protein engineering method analysis of 17 mutants reveals that the transition state of the reaction resembles that for the folding of the intact protein. The transition state for the binding of the fragment has a single alpha-helix with a fully formed N-terminal region. The individual fragments are nonnative, with regions of nonnative buried hydrophobic clusters but lacking native secondary structure.

The mechanism of the association of fragments of the chymotrypsin inhibitor-2 involves two steps, a major second-order phase and a slow first order phase. The association and folding of the fragments takes place in the second-order step and a cis-trans peptidyl-prolyl isomerization in one fragment results in the first-order step. The CI-2 fragments exhibit tight association despite the absence of clear initiation sites for the association.

The article studies the complete thermodynamic description of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) by calorimetric and spectroscopic technique. Findings suggest the survival of a low-energy barrier among the different macrostates of hNedd4-WW4.

We investigated the role of electrostatic charges at positions D72 and K8 in the function and structural stability of cytochrome c6 from Nostoc sp. PCC 7119 (cyt c6). A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions. All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea-induced folding equilibrium relaxation experiments and differential scanning calorimetry. Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I. This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I. Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein. D72 apparently participates in electrostatic interactions that stabilize the structure of cyt c6. The destabilizing effect was reduced when aspartate was replaced by the small amino acid alanine. Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild-type under all tested conditions. [ABSTRACT FROM AUTHOR]

Studies the association kinetics involved in two chymotrypsin inhibitor-2 fragments generated by cyanogen bromide cleavage. Cooperative folding phase; Time course for structure formation; Rate constants.