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1. Effects of Protein Unfolding on Aggregation and Gelation in Lysozyme Solutions

   https://doi.org/10.3390/biom10091262  

   Nikfarjam, Shakiba; Jouravleva, Elena V.; Anisimov, Mikhail A.; Woehl, Taylor J. (September 2020, Biomolecules)

   null (Ed.)

   In this work, we investigate the role of folding/unfolding equilibrium in protein aggregation and formation of a gel network. Near the neutral pH and at a low buffer ionic strength, the formation of the gel network around unfolding conditions prevents investigations of protein aggregation. In this study, by deploying the fact that in lysozyme solutions the time of folding/unfolding is much shorter than the characteristic time of gelation, we have prevented gelation by rapidly heating the solution up to the unfolding temperature (~80 °C) for a short time (~30 min.) followed by fast cooling to the room temperature. Dynamic light scattering measurements show that if the gelation is prevented, nanosized irreversible aggregates (about 10–15 nm radius) form over a time scale of 10 days. These small aggregates persist and aggregate further into larger aggregates over several weeks. If gelation is not prevented, the nanosized aggregates become the building blocks for the gel network and define its mesh length scale. These results support our previously published conclusion on the nature of mesoscopic aggregates commonly observed in solutions of lysozyme, namely that aggregates do not form from lysozyme monomers in their native folded state. Only with the emergence of a small fraction of unfolded proteins molecules will the aggregates start to appear and grow. 

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2. Distribution and solvent exposure of Hsp70 chaperone binding sites across the Escherichia coli proteome

   https://doi.org/10.1002/prot.26456  

   Chen, Xi; Hutchinson, Rachel B.; Cavagnero, Silvia (January 2023, Proteins: Structure, Function, and Bioinformatics)

   Abstract Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding‐site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client‐protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome‐wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2258Escherichia coli(E. coli) proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent‐exposed than other nonpolar sites, in protein native states. Our findings show that the majority ofE. coliproteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70‐bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70‐chaperone‐binding traits have evolved to favor Hsp70‐assisted protein folding devoid of aggregation. 

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3. Cataract-prone variants of γD-crystallin populate a conformation with a partially unfolded N-terminal domain under native conditions

   https://doi.org/10.1073/pnas.2410860122  

   Volz, Sara; Malone, Jadyn_R; Guseman, Alex_J; Gronenborn, Angela_M; Marqusee, Susan (February 2025, Proceedings of the National Academy of Sciences)

   Human γD-crystallin, a monomeric protein abundant in the eye lens nucleus, must remain stably folded for an individual’s entire lifetime to avoid aggregation and protein deposition-associated cataract formation. γD-crystallin contains two homologous domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), which interact via a hydrophobic interface. Several familial mutations in the gamma crystallin gene are linked to congenital early-onset cataract, most of which affect the NTD. Some of these, including V75D and W42R, are known to populate intermediates under partially denaturing conditions possessing a natively folded CTD and a completely unfolded NTD. We employed hydrogen–deuterium exchange mass spectrometry to probe the structural and energetic features of variants of γD-crystallin under both native and partially denaturing conditions. For V75D and W42R, we identify a species under native conditions that retains partial structure in the NTD and is structurally and energetically distinct from the intermediate populated under partially denaturing conditions. Residues at the NTD–CTD interface play crucial roles in stabilizing this intermediate, and disruption of interface contacts either by amino acid substitution or partial denaturation permits direct observation of two intermediates simultaneously. These data suggest that the intermediate identified under native conditions is accessed from the native state and not on the folding pathway. The intermediate we have identified here exposes hydrophobic amino acids that are buried in both the folded full-length protein and in the protein’s stable isolated domains. Such nonnative exposure of a hydrophobic patch may play an important role in cataract formation. 

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4. Implications of the unfolded state in the folding energetics of heterogeneous-backbone protein mimetics

   https://doi.org/10.1039/D2SC04427G  

   Santhouse, Jacqueline R.; Leung, Jeremy M.; Chong, Lillian T.; Horne, W. Seth (October 2022, Chemical Science)

   Sequence-encoded folding is the foundation of protein structure and is also possible in synthetic chains of artificial chemical composition. In natural proteins, the characteristics of the unfolded state are as important as those of the folded state in determining folding energetics. While much is known about folded structures adopted by artificial protein-like chains, corresponding information about the unfolded states of these molecules is lacking. Here, we report the consequences of altered backbone composition on the structure, stability, and dynamics of the folded and unfolded states of a compact helix-rich protein. Characterization through a combination of biophysical experiments and atomistic simulation reveals effects of backbone modification that depend on both the type of artificial monomers employed and where they are applied in sequence. In general, introducing artificial connectivity in a way that reinforces characteristics of the unfolded state ensemble of the prototype natural protein minimizes the impact of chemical changes on folded stability. These findings have implications in the design of protein mimetics and provide an atomically detailed picture of the unfolded state of a natural protein and artificial analogues under non-denaturing conditions. 

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5. Electrical unfolding of cytochrome c during translocation through a nanopore constriction

   https://doi.org/10.1073/pnas.2016262118  

   Tripathi, Prabhat; Benabbas, Abdelkrim; Mehrafrooz, Behzad; Yamazaki, Hirohito; Aksimentiev, Aleksei; Champion, Paul M.; Wanunu, Meni (April 2021, Proceedings of the National Academy of Sciences)

   null (Ed.)

   Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field–driven translocation behavior of cytochrome c (cyt c ) through ultrathin silicon nitride (SiN x ) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field–induced deformability. 

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