An official website of the United States government
The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins, combined with the exposure of their residues, accounts for this sensitivity. One context in which IDPs play important roles that are concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat-soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet the mechanisms underlying this synergy differ between IDP families.
more »
« less
This content will become publicly available on December 1, 2026
Single-molecule techniques for probing the conformation, dynamics, and interactions of intrinsically disordered proteins
Intrinsically disordered proteins (IDPs) rely on inherent conformational dynamics to support vital cellular functions such as signaling and regulation. The heterogeneity of the conformational ensembles of IDPs poses a major challenge for classical biophysical techniques such as nuclear magnetic resonance, small-angle x-ray scattering, and circular dichroism, which measure ensemble-averaged properties and are unable to resolve the full range of substates sampled by IDPs or their interconversions. To address these limitations of ensemble-based methods, single-molecule techniques are essential to resolve the conformational heterogeneity and dynamics by continuously monitoring various properties of individual IDPs. In this review, we focus on the most commonly employed single-molecule techniques, briefly describe their methodologies, and highlight recent developments and applications of each technique toward the study of IDP structure, dynamics and interactions.
more »
« less
- Award ID(s):
- 2516941
- PAR ID:
- 10681318
- Publisher / Repository:
- AIP Publishing
- Date Published:
- Journal Name:
- Biophysics Reviews
- Volume:
- 6
- Issue:
- 4
- ISSN:
- 2688-4089
- Subject(s) / Keyword(s):
- biophysics
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results demonstrate that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.more » « less
-
Abstract The preponderance of intrinsically disordered proteins (IDPs) in the eukaryotic proteome, and their ability to interact with each other, and with folded proteins, RNA, and DNA for functional purposes, have made it important to quantitatively characterize their biophysical properties. Toward this end, we developed the transferable self‐organized polymer (SOP‐IDP) model to calculate the properties of several IDPs. The values of the radius of gyration () obtained from SOP‐IDP simulations are in excellent agreement (correlation coefficient of 0.96) with those estimated from SAXS experiments. For AP180 and Epsin, the predicted values of the hydrodynamic radii () are in nearly quantitative agreement with those from fluorescence correlation spectroscopy (FCS) experiments. Strikingly, the calculated SAXS profiles for 36 IDPs are also nearly superimposable on the experimental profiles. The dependence of and the mean end‐to‐end distance () on chain length, , follows Flory's scaling law, ( and ), suggesting that globally IDPs behave as synthetic polymers in a good solvent. This finding depends on the solvent quality, which can be altered by changing variables such as pH and salt concentration. The values of and are 0.20 and 0.48 nm, respectively. Surprisingly, finite size corrections to scaling, expected on theoretical grounds, are negligible for and . In contrast, only by accounting for the finite sizes of the IDPs, the dependence of experimentally measurable on can be quantitatively explained using . Although Flory scaling law captures the estimates for , , and accurately, the spread of the simulated data around the theoretical curve is suggestive of of sequence‐specific features that emerge through a fine‐grained analysis of the conformational ensembles using hierarchical clustering. Typically, the ensemble of conformations partitions into three distinct clusters, having different equilibrium populations and structural properties. Without any further readjustments to the parameters of the SOP‐IDP model, we also obtained nearly quantitative agreement with paramagnetic relaxation enhancement (PRE) measurements forα‐synuclein. The transferable SOP‐IDP model sets the stage for several applications, including the study of phase separation in IDPs and interactions with nucleic acids.more » « less
-
de Groot, Bert L. (Ed.)Intrinsically disordered proteins (IDPs) are highly dynamic systems that play an important role in cell signaling processes and their misfunction often causes human disease. Proper understanding of IDP function not only requires the realistic characterization of their three-dimensional conformational ensembles at atomic-level resolution but also of the time scales of interconversion between their conformational substates. Large sets of experimental data are often used in combination with molecular modeling to restrain or bias models to improve agreement with experiment. It is shown here for the N-terminal transactivation domain of p53 (p53TAD) and Pup, which are two IDPs that fold upon binding to their targets, how the latest advancements in molecular dynamics (MD) simulations methodology produces native conformational ensembles by combining replica exchange with series of microsecond MD simulations. They closely reproduce experimental data at the global conformational ensemble level, in terms of the distribution properties of the radius of gyration tensor, and at the local level, in terms of NMR properties including 15 N spin relaxation, without the need for reweighting. Further inspection revealed that 10–20% of the individual MD trajectories display the formation of secondary structures not observed in the experimental NMR data. The IDP ensembles were analyzed by graph theory to identify dominant inter-residue contact clusters and characteristic amino-acid contact propensities. These findings indicate that modern MD force fields with residue-specific backbone potentials can produce highly realistic IDP ensembles sampling a hierarchy of nano- and picosecond time scales providing new insights into their biological function.more » « less
-
Abstract Mechanical modulation of biomolecular structures by single‐molecule techniques, such as optical tweezers, has revealed subtle conformational dynamics and enabled precise modulation of functional properties. However, such tools are limited to manipulating one or a few molecules at a time in extracellular settings, posing significant challenges for scaling force‐based methods to achieve high sensitivity and efficacy both outside and within cells. Here, low‐power (<5.3 mW cm−2) ultrasound is employed to generate sono‐mechanical forces without formation of sonodynamic radicals, which are known to irreversibly alter molecular structures. By calibrating against optical‐tweezers‐based single‐molecule force spectroscopy, this study quantifies for the first time that at least 29 pN sono‐mechanical force can be generated at 5.3 mW cm−2sonication power, capable of simultaneously and reversibly unfold an ensemble set of DNA structures, including G‐quadruplexes and hairpins. Notably, the same sono‐mechanical unfolding is observed in cells, where intercalated doxorubicin ligands are released from unfolded DNA hairpin carriers, resulting in targeted cancer cell death. These findings show ultrasound can simultaneously manipulate a large population of biomolecules without requiring fixed orientations, offering a flexible and nonintrusive force generation to reversibly unfold molecular structures. This platform holds profound potential for applications in molecular biophysics, smart materials, and precision medicines.more » « less
