Reduced‐denatured ribonuclease A is not in a compact state

Nöppert, Angela; Gast, Klaus; Müller-Frohne, Marlies; Zirwer, Dietrich; Damaschun, G.

doi:10.1016/0014-5793(96)00048-8

Cited by 51 publications

(73 citation statements)

References 19 publications

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“…[40][41][42][43] The SAXS data recorded on ACTR and hNHE1cdt show that an overall contraction of the ensemble of structures occurs with increasing temperature. The contraction could be caused by a change in either the transient secondary structure or the tertiary structure.…”

Section: Discussionmentioning

confidence: 98%

Temperature‐dependent structural changes in intrinsically disordered proteins: Formation of α‒helices or loss of polyproline II?

Kjærgaard¹,

Nørholm²,

et al. 2010

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Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient a-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.Keywords: intrinsically disordered protein (IDP); transient secondary structure; temperature dependence; polyproline II; circular dichroism (CD); nuclear magnetic resonance spectroscopy (NMR); sodium-proton (Na1/H1) exchanger 1 (NHE1); S-phase delayed 1 (Spd1); activator for thyroid hormone and retinoid receptors (ACTR) Abbreviations: ACTR, activator for thyroid hormone and retinoid receptors; CBP, CREB-binding protein; CD, circular dichroism; IDP, intrinsically disordered protein; NCBD, nuclear coactivator binding domain; NHE1cdt, sodium-proton (Na þ /H þ ) exchanger 1 C-terminal distal tail; NMR, nuclear magnetic resonance; PPII, polyproline II; RDC, residual dipolar coupling; SAXS, small-angle X-ray scattering; Spd1, S-phase delayed 1.

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Section: Discussionmentioning

confidence: 98%

Temperature‐dependent structural changes in intrinsically disordered proteins: Formation of α‒helices or loss of polyproline II?

Kjærgaard¹,

Nørholm²,

et al. 2010

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“…First, we used a two-state folder without disulfides to avoid ambiguities from the influence of structured intermediates or disulfides on the properties of the denatured state. Second, we used single-molecule FRET to monitor the unfolded state even under conditions where the folded state is populated, avoiding the need for additional destabilization by reduction or low pH (35,37,39,40). Third, aggregation does not occur at the exceedingly low concentrations used in single-molecule experiments and can be monitored in situ (60).…”

Section: Discussionmentioning

confidence: 99%

“…For simple polymers and polymer models that do not involve a temperature-dependent interaction energy, conformational entropy favors open conformations, leading to chain expansion with increasing temperature (34), an effect that is assumed frequently to be dominant also for unfolded proteins. Although none of the sparse experimental results on this topic show such an expansion, they exhibit substantial variation, ranging from the absence of any detectable temperature dependence (35,36) to slight (37,38) and stronger (39) collapse, with some inconsistencies between measurements even on the same protein (35,37,40), calling for a systematic investigation of this issue. Ensemble investigations are limited largely to highly unfolding conditions to exclude interference from the signal of folded molecules and to minimize aggregation.…”

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confidence: 99%

Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins

Nettels

Müller-Späth²,

Küster³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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216

252

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We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With singlemolecule FRET, this question can be addressed even under nearnative conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperaturedependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin ␣ suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.FRET ͉ polymer ͉ protein folding ͉ secondary structure ͉ chain dimensions T here is an increasing interest in the properties of unfolded proteins and their roles in the folding and cellular functions of proteins. A key motivation is that many proteins are marginally stable and only fold in the presence of their ligands or binding partners, opening new regulatory possibilities (1, 2). An important reason for recent progress is the growing availability of methods that provide structural information on these conformationally heterogeneous systems, such as NMR (3), scattering methods (4, 5), and single-molecule . Although NMR provides mostly local details, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and single-molecule FRET provide overall hydrodynamic or long-range distance information. An important advantage of single-molecule FRET is the separation of folded and unfolded subpopulations (9). As a result, unfolded state properties can be investigated even in the presence of folded molecules (i.e., under near-native conditions, which are physiologically most relevant). This advance has led to the observation of a continuous collapse of the unfolded state with decreasing denaturant concentrations (10), a behavior that now has been demonstrated for a large number of proteins (11-18) and peptides (19). Recent advances in the application of theoretical models have led to a quantitative description of this unfolded state collapse in terms of polymerphysical concepts (15,(20)(21)(22)(23). Such chain compaction also has been demonstrated to result in increased internal friction and a slowdown of intramolecular dynamics of the polypeptide (19, 26), which can affect...

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“…Studying the structure, dynamics and function of these disordered polypeptide chains, as well as other disordered biopolymers, requires new experimental methods, and new methods of interpreting the data, since they are inherently averaged over a broad ensemble of heteropolymer configurations [2][3][4] . Examples of experiments which can help address this challenge include: nuclear magnetic resonance (NMR), which can provide short-range structural information via scalar coupling, chemical shift and NOE data 5 , as well as long-range information by paramagnetic relaxation enhancement (PRE) measurements 6 , and global information from diffusion coefficient measurements; light-scattering 7 and two-focus FCS 4,8 , which also yield diffusion coefficients and hence hydrodynamic radius; small-angle X-ray (or neutron) scattering (SAXS or SANS), which directly gives information on inter-and intramolecular pair distance distributions 9 ; and Förster resonance energy transfer (FRET), in particular single-molecule FRET, which probes the distribution of distances between pairs of chromophore labels attached to the molecule of interest, as well as the associated dynamics, and often enables structured and unstructured subpopulations to be separated 10,11 . Obtaining as detailed as possible a picture of the disordered ensemble would ideally combine information from all of these experiments, if available.…”

Section: Introductionmentioning

confidence: 99%

Inferring Properties of Disordered Chains From FRET Transfer Efficiencies

Zheng

Zerze

Borgia

et al. 2018

Biophysical Journal

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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...

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Reduced‐denatured ribonuclease A is not in a compact state

Cited by 51 publications

References 19 publications

Temperature‐dependent structural changes in intrinsically disordered proteins: Formation of α‒helices or loss of polyproline II?

Temperature‐dependent structural changes in intrinsically disordered proteins: Formation of α‒helices or loss of polyproline II?

Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins

Inferring Properties of Disordered Chains From FRET Transfer Efficiencies

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