Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water

Riback, Joshua A.; Bowman, Micayla A.; Zmyslowski, Adam M.; Knoverek, Catherine R.; Jumper, John; Hinshaw, James R.; Kaye, Emily B.; Freed, Karl F.; Clark, Patricia L.; Sosnick, Tobin R.

doi:10.1126/science.aan5774

Cited by 206 publications

(307 citation statements)

References 39 publications

Supporting

Mentioning

279

Contrasting

Unclassified

Order By: Relevance

“…Application of polymer-theoretic values of G to the smFRET inferred R ee results in R g 24-27Å for Sic1 and 26-29Å for pSic1 (SI Appendix Table S3 ). These inferred R g values are systematically smaller than those inferred from the SAXS dataset (see below), or from integrated ensemble modelling (see below), similar to previously reported discrepancies between smFRET and SAXS [27,34,35]. A model of chain statistics does not need to be specified, however, these methods are limited in describing IDPs and unfolded proteins [35,36].…”

Section: Introductionsupporting

confidence: 82%

“…Similarly, Riback and coworkers have recently introduced a procedure for fitting SAXS data by pregenerating ensembles of conformations with different properties (specifically, the strength and patterning of inter-residue attractions) and extracting dimensionless "molecular form factors" (MFFs) [34,38]. The properties of interest are then inferred from the ensemble whose MFF best fits the data.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

Gomes

Krzeminski

Namini

et al. 2020

Preprint

View full text Add to dashboard Cite

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging. Transient long-range interactions in IDPs are known to have important functional implications. Thus, in order to calculate reliable structural ensembles of IDPs, the data used in their calculation must capture these important structural features. We use integrative modelling to understand and implement conformational restraints imposed by the most common structural techniques for IDPs: NMR spectroscopy, small-angle X-ray scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Using the disordered N-terminal region of the Sic1 protein as a test case, we find that only Paramagnetic Relaxation Enhancement (PRE) and smFRET measurements are able to unambiguously report on transient long-range interactions. It is precisely these features which lead to deviations from homopolymer statistics and divergent structural inferences in non-integrative sm-FRET and SAXS analysis. Furthermore, we find that the sequence-specific deviations from homopolymer statistics are consistent with biophysical models of Sic1 function that are mediated by phospho-sensitive binding to its partner Cdc4. To our knowledge, these are the first conformational ensembles for an IDP in physiological conditions that are simultaneously consistent with smFRET, SAXS, and NMR data.Our results stress the importance of integrating the global and local structural information provided by SAXS and Chemical Shifts, respectively, with information on specific inter-residue distances from PRE and smFRET. Our integrative modelling approach and quantitative polymer-physics-based characterization of the experimentally-restrained ensembles could be used to implement a rigorous taxonomy for the description and classification of IDPs as heteropolymers. Significance StatementIntrinsically disordered proteins (IDPs) exhibit highly dynamic and heterogeneous conformations, which impedes rigorous structural characterization and understanding of their biological functions. Sic1 regulates the yeast cell cycle through phospho-sensitive binding to its partner Cdc4 and is paradigmatic of IDPs that bind tightly without partial/transient folding. In this paper, we integrated new and existing structural data from nuclear magnetic resonance, small-angle X-ray scattering and single-molecule fluorescence to calculate conformational ensembles for Sic1 and its phosphorylated state, pSic1. Data mining of these ensembles reveal unique features distinguishing Sic1/pSic1 from homopolymer statistics, such as overall compactness and large end-to-end distance fluctuations. Integrating experiments probing disparate scales, computational modelling, and polymer physics provides new and valuable insights into the conformation-to-function relationships in IDPs.

show abstract

Section: Introductionsupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

Gomes

Krzeminski

Namini

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…After submission of our work, a novel method of analyzing small-angle X-ray scattering (SAXS) experiments was published which also allows a scaling exponent to be determined from a single experiment 75 . An approach similar to the SAW- model should also be applicable to analyzing SAXS experiments to obtain both the scaling exponent and Rg.…”

Section: Discussionmentioning

confidence: 99%

Inferring Properties of Disordered Chains From FRET Transfer Efficiencies

Zheng

Zerze

Borgia

et al. 2018

Biophysical Journal

View full text Add to dashboard Cite

Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely, the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble properties from FRET data, in which the polymer scaling exponent is allowed to vary with solution conditions. In its simplest form, it requires either the mean FRET efficiency or fluorescence lifetime information. In order to test the accuracy of the method, we have utilized both synthetic FRET data from implicit and explicit solvent simulations for 30 different protein sequences, and experimental single-molecule FRET data for an intrinsically disordered and a denatured protein. In all cases, we find that the inferred radii of gyration are within 10% of the true values, thus providing higher accuracy than simpler polymer models. In addition, the scaling exponents obtained by our procedure are in good agreement with those determined directly from the molecular ensemble. Our approach can in principle be generalized to treating other ensemble-averaged functions of intramolecular distances from experimental data. Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment, so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble proper...

show abstract

“…Buffer subtraction, Guinier analysis and Kratky transformation was performed in Matlab (Mathworks). Final data was also fit to an empirically derived molecular form factor for unfolded proteins [31].…”

Section: Measurement Of Binding Affinitiesmentioning

confidence: 99%

Protein network structure enables switching between liquid and gel states

Schmit

Bouchard

Martin

et al. 2019

Preprint

View full text Add to dashboard Cite

Biomolecular condensates are emerging as an important organizational principle within living cells. These condensed states are formed by phase separation, yet little is known about how material properties are encoded within the constituent molecules and how the specificity for being in different phases is established. Here we use analytic theory to explain the phase behavior of the cancer-related protein SPOP and its substrate DAXX. Binary mixtures of these molecules have a phase diagram that contains dilute liquid, dense liquid, and gel states. We show that these discrete phases appear due to a competition between SPOP-DAXX and DAXX-DAXX interactions. The stronger SPOP-DAXX interactions dominate at sub-stoichiometric DAXX concentrations leading to the formation of crosslinked gels. The theory shows that the driving force for gel formation is not the binding energy, but rather the entropy of distributing DAXX molecules on the binding sites. At high DAXX concentrations the SPOP-DAXX interactions saturate, which leads to the dissolution of the gel and the appearance of a liquid phase driven by weaker DAXX-DAXX interactions.This competition between interactions allows multiple dense phases to form in a narrow region of parameter space. We propose that the molecular architecture of phase-separating proteins governs the internal structure of dense phases, their material properties and their functions. Analytical theory can reveal these properties on the long length and time scales relevant to biomolecular condensates.

show abstract

Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water

Cited by 206 publications

References 39 publications

Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

Inferring Properties of Disordered Chains From FRET Transfer Efficiencies

Protein network structure enables switching between liquid and gel states

Contact Info

Product

Resources

About