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1. A modular approach to map out the conformational landscapes of unbound intrinsically disordered proteins

   https://doi.org/10.1073/pnas.2113572119  

   Luong, Thinh D.; Nagpal, Suhani; Sadqi, Mourad; Muñoz, Victor (June 2022, Proceedings of the National Academy of Sciences)

   Intrinsically disordered proteins (IDPs) fold upon binding to select/recruit multiple partners, morph around the partner's structure, and exhibit allostery. However, we do not know whether these properties emerge passively from disorder, or rather are encoded into the IDP's folding mechanisms. A main reason for this gap is the lack of suitable methods to dissect the energetics of IDP conformational landscapes without partners. Here we introduce such an approach that we term molecular LEGO, and apply it to NCBD, a helical, molten globule–like IDP, as proof of concept. The approach entails the experimental and computational characterization of the protein, its separate secondary structure elements (LEGO building blocks), and their supersecondary combinations. Comparative analysis uncovers specific, yet inconspicuous, energetic biases in the conformational/folding landscape of NCBD, including 1) strong local signals that define the three native helices, 2) stabilization of helix–helix interfaces via soft pairwise tertiary interactions, 3) cooperative stabilization of a heterogeneous three-helix bundle fold, and 4) a dynamic exchange between sets of tertiary interactions (native and nonnative) that recapitulate the different structures NCBD adopts in complex with various partners. Crucially, a tug of war between sets of interactions makes NCBD gradually shift between structural subensembles as a conformational rheostat. Such conformational rheostatic behavior provides a built-in mechanism to modulate binding and switch/recruit partners that is likely at the core of NCBD's function as transcriptional coactivator. Hence, the molecular LEGO approach emerges as a powerful tool to dissect the conformational landscapes of unbound IDPs and rationalize their functional mechanisms. 

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2. 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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3. In silico protein dynamics in the human cytoplasm: Partial folding, misfolding, fold switching, and non‐native interactions

   https://doi.org/10.1002/pro.4790  

   Russell, Premila P. Samuel; Rickard, Meredith M.; Boob, Mayank; Gruebele, Martin; Pogorelov, Taras V. (October 2023, Protein Science)

   Abstract We examine the influence of cellular interactions in all‐atom models of a section of theHomo sapienscytoplasm on the early folding events of the three‐helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30‐microsecond simulations. We did however capture the formation of all three helices in a compact native‐like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta‐sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild‐type sequence. This study demonstrates that even fast‐folding proteins can get stuck in non‐native states in the cell, making them useful models for protein–chaperone interactions and early stages of aggregate formation relevant to cellular disease. 

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4. Zinc Alters the Supramolecular Organization of Nucleic Acid Complexes with Full-Length TIA1

   Yizhuo Yang, Keith J. (January 2023, bioRxiv)

   na (Ed.)

   T-Cell Intracellular Antigen-1 (TIA1) is a 43 kDa multi-domain RNA-binding protein involved in stress granule formation during eukaryotic stress response, and has been implicated in neurodegenerative diseases including Welander distal myopathy and amyotrophic lateral sclerosis. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids and a C-terminal Q/N-rich prion-related domain (PRD) which has been variously described as intrinsically disordered or prion inducing and is believed to play a role in promoting liquid-liquid phase separation connected with the assembly of stress granule formation. Motivated by the fact that our prior work shows RRMs 2 and 3 are well-ordered in an oligomeric full-length form, while RRM1 and the PRD appear to phase separate, the present work addresses whether the oligomeric form is functional and competent for binding, and probes the consequences of nucleic acid binding for oligomerization and protein conformation change. New SSNMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at the RRM2 domain, but also weakly at the RRM3 domain, and Zn2+ binds primarily to RRM3. Binding of Zn2+ and DNA was reversible for the full-length wild type oligomeric form, and did not lead to formation of amyloid fibrils, despite the presence of the C-terminal prion-related domain. While TIA1:DNA complexes appear as long “daisy chained” structures, the addition of Zn2+ caused the structures to collapse. We surmise that this points to a regulatory role for Zn2+. By occupying various “half” binding sites on RRM3 Zn2+ may shift the nucleic acid binding off RRM3 and onto RRM2. More importantly, the use of different half sites on different monomers may introduce a mesh of crosslinks in the supramolecular complex rendering it compact and markedly reducing the access to the nucleic acids (including transcripts) from solution. 

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5. Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy

   https://doi.org/10.1073/pnas.2020083118  

   Jacobson, David R.; Perkins, Thomas T. (March 2021, Proceedings of the National Academy of Sciences)

   Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔGfor mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔGfor mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val²¹⁷and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔGfor a fully folded membrane protein embedded in its native bilayer. 

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