The heat-shock factor Hsp70 and other molecular chaperones play a central role in nascent protein folding. Elucidating the task performed by individual chaperones within the complex cellular milieu, however, has been challenging. One strategy for addressing this goal has been to monitor protein biogenesis in the absence and presence of inhibitors of a specific chaperone, followed by analysis of folding outcomes under both conditions. In this way, the role of the chaperone of interest can be discerned. However, development of chaperone inhibitors, including well-known proline-rich antimicrobial peptides, has been fraught with undesirable side effects, including decreased protein expression yields. Here, we introduce KLR-70, a rationally designed cationic inhibitor of the Escherichia coli Hsp70 chaperone (also known as DnaK). KLR-70 is a 14-amino acid peptide bearing naturally occurring residues and engineered to interact with the DnaK substrate-binding domain. The interaction of KLR-70 with DnaK is enantioselective and is characterized by high affinity in a buffered solution. Importantly, KLR-70 does not significantly interact with the DnaJ and GroEL/ES chaperones, and it does not alter nascent protein biosynthesis yields across a wide concentration range. Some attenuation of the anti-DnaK activity of KLR-70, however, has been observed in the complex E. coli cell-free environment. Interestingly, the d enantiomer D-KLR-70, unlike its all-L KLR-70 counterpart, does not bind the DnaK and DnaJ chaperones, yet it strongly inhibits translation. This outcome suggests that the two enantiomers (KLR-70 and D-KLR-70) may serve as orthogonal inhibitors of chaperone binding and translation. In summary, KLR-70 is a novel chaperone inhibitor with high affinity and selectivity for bacterial Hsp70 and with considerable potential to help in parsing out the role of Hsp70 in nascent protein folding.

Anfinsen's thermodynamic hypothesis does not explicitly take into account the possibility of protein aggregation. Here, we introduce a cyclic-perturbation approach to prove that not only the native state but also soluble aggregates of most proteins can be highly populated under mild, physiologically relevant conditions, even at very low concentration. Surprisingly, these aggregates are not necessarily amyloid in nature and are usually not observed in bioactive proteins due to the extremely low kinetic flux from the native state toward a region of the chemical-potential landscape encoding aggregates. We first illustrate this concept for the representative model protein apomyoglobin-at room temperature and no denaturant-and demonstrate kinetic trapping of the native state relative to at least two different types of soluble, predominantly nonamyloid aggregates. The concentration and temperature dependence of aggregation confirm the above scenario. Extension of our analysis to the Escherichia coli proteome shows that the majority of the soluble bacterial proteome is also kinetically trapped in the nonaggregated state. Hence, the existence and low kinetic accessibility of large aggregates at room temperature and pH 6-7 is a general phenomenon. We also show that the average critical protein concentration for aggregation of most of the bacterial proteome is extremely small, much lower than the typical cellular protein concentration. Hence, the thermodynamic driving force for protein aggregation is large even if aggregation does not usually occur in healthy cells due to kinetic trapping. A broader view of Anfinsen's thermodynamic hypothesis encompassing all protein states, including aggregates, is necessary to understand the behavior of proteins in their natural environment.

The transcriptional factor, c-Myb R2R3, is minimum unit for DNA-binding and shows largely flexible conformation in solution, which is important for specific DNA-binding function. Here we investigated the structural dynamics of c-Myb R2R3 induced by the DNA-binding, using circular dichroism (CD), diffracted X-ray tracking (DXT), and isothermal titration calorimetry (ITC). DXT is recently developed methods and can evaluate the protein structural fluctuations by detecting the movement of a gold-nanocrystal attached to the target protein. Thermal stability of R2R3 was increased in the presence of cognate DNA, suggesting that the structure was changed in more rigid upon the DNA-binding. The resultant curve of the mean square angular displacements (MSD) obtained from DXT clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using ITC. The results of the MSD curves also indicate that the translational length reduces by approximately half upon DNA binding.

Although the role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, many disease-associated mutations in IDPs are charge-neutral. Earlier, we studied the effects of the disease-associated Val66Met substitution at the midpoint of the prodomain of precursor brain-derived neurotrophic factor (proBDNF) using fully atomistic molecular dynamics simulations. Val66Met substitution is found in 25% of the American population, which has been widely studied for its association with aging-related and stress-related disorders, reduced volume of the hippocampus, and variations in episodic memory. We found that the local secondary structure, transient tertiary contacts, and compactness of the protein are correlated to backbone configuration around residue 66. The midpoint location and the substitution at the most highly charged region of the protein played a critical role in causing the conformational changes of Val66Met substitution. To gain further insight into the generalizability of the found mechanism with which a hydrophobic-tohydrophobic substitution can impact the IDP's conformational ensemble and to further establish the significance of substitution location, we studied 5 more hydrophobic substitutions at residue 66. We report on fullyatomistic temperature replica exchange molecular dynamics simulations of the 90 residue proBDNF for Ala66, Ile66, Leu66, Phe66 and Tyr66 sequence. Analyzing and comparing the residue level insight from all 5 simulations helped us in further establishing the significance of charge-neutral mutations in IDPs.