Macromolecular Crowding in the Escherichia coli Periplasm Maintains α-Synuclein Disorder

McNulty, Brian C.; Young, Gregory B.; Pielak, Gary J.

doi:10.1016/j.jmb.2005.11.033

Cited by 163 publications

(182 citation statements)

References 19 publications

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“…Resonance assignments were made using standard methods [HNCO, HN(CO)CA, HNCA, HNCACB, 15 N-edited NOESY, and TOCSY]. A comparison of our assignments with those made for αSyn upon association with lipid (which drives helix formation) shows somewhat decreased chemical shift dispersion in the present case, indicating that helix formation is dynamic (15,16 (17). Chemical shift-based secondary structural analysis using TALOS+ (18) indicates that with the exception of short segments near the N terminus of the polypeptide, the structure of the peptide is dynamic (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 74%

A soluble α-synuclein construct forms a dynamic tetramer

Wang

Perovic

Chittuluru

et al. 2011

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

435

522

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A heterologously expressed form of the human Parkinson diseaseassociated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 Nterminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three-or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein. T he protein α-synuclein (αSyn) is associated with the two most prevalent neurodegenerative diseases, Parkinson disease (PD) and Alzheimer's disease (AD). The presence of αSyn-rich aggregates (Lewy bodies) in neurons of the substantia nigra is the defining histopathological hallmark of PD, and is used to differentiate PD from other neurological disorders (1). Monogenic point mutations (A30P, A53T, and E46K) as well as gene duplication and triplication of the αSyn locus have been identified as causal factors of early onset familial PD; E46K has also been associated with Lewy body dementia, the second most common form of dementia after AD (2-4).αSyn is small (140 residues), and though the C-terminal region (∼residues 100-140) is highly acidic and expected to be disordered, the first 100 residues are predicted to be structured and to have α-helical propensity (SI Appendix, Fig. S1). Stable helical structures have been detected by circular dichroism (CD) and NMR when αSyn is incubated with detergent micelles and lipid vesicles (5, 6). Soluble αSyn is typically referred to as an "intrinsically disordered" protein (7,8). However, we herein report the biophysical characterization of a purified soluble form of αSyn that is oligomeric and fractionally occupies helical structures in the absence of micelles or vesicles. The αSyn construct used in our work is purified by use of an N-terminal GST affinity tag under mild conditions to preserve any native structure. After removal of the GST tag, a 10-residue N-terminal extension remains on the αSyn. However, the similarity of the 1 H, 15 N heteronuclear single-quantum coherence (HSQC) fingerprint of our αSyn construct (SI Appendix, Figs. S2 and S3) to those reported by other groups for αSyn suggests that the N-terminal extension does not change structural tendencies significantly. The αSyn construct described here is not toxic to membranes or cells, does not readily aggregate or ...

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Section: Resultsmentioning

confidence: 74%

A soluble α-synuclein construct forms a dynamic tetramer

Wang

Perovic

Chittuluru

et al. 2011

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

435

522

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“…Overexpressed α-synuclein is found in the periplasm of E. coli [56]. Using in-cell NMR spectroscopy, McNulty et al [57] investigated the conformations of α-synuclein in the periplasm of E. coli. They found that α-synuclein adopts a more compact form than is observed in a dilute solution.…”

Section: Protein Folding In Cellular Environmentsmentioning

confidence: 99%

Protein folding in confined and crowded environments

Zhou

2008

Archives of Biochemistry and Biophysics

151

137

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Confinement and crowding are two major factors that can potentially impact protein folding in cellular environments. Theories based on considerations of excluded volumes predict disparate effects on protein folding stability for confinement and crowding: confinement can stabilize proteins by over 10k B T but crowding has a very modest effect on stability. On the other hand, confinement and crowding are both predicted to favor conformations of the unfolded state which are compact, and consequently may increase the folding rate. These predictions are largely borne out by experimental studies of protein folding under confined and crowded conditions in the test tube. Protein folding in cellular environments is further complicated by interactions with surrounding surfaces and other factors. Concerted theoretical modeling and test-tube and in vivo experiments promise to elucidate the complexity of protein folding in cellular environments.The complexity of the cellular environments in which proteins function is increasingly been appreciated [1]. Potential impacts of two related, but distinct factors, confinement and macromolecular crowding, on protein folding are receiving extensive attention [2][3][4][5]. Confinement arises from encapsulation of proteins in compartments with dimensions on the scales of 10's to 100's Å, which are only moderately larger than the sizes of the proteins themselves. Examples of such compartments include the Anfinsen cage of chaperonins and the exit tunnel of ribosomes. Crowding refers to the exclusion of volumes to proteins of interest by the presence of other macromolecules. The aims of this paper are to discuss potential impacts of confinement and crowding on protein folding and to review the progress made by recent studies on protein folding in confined and crowded conditions and in cellular environments.

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“…Given their lack of persistent structure, the conformations of IDPs are expected to be particularly sensitive to the effects of such molecular crowding. Indeed, first experiments indicate that some IDPs gain structure upon crowding (15), whereas others do not (16)(17)(18), but may change their dimensions (19)(20)(21). The question of how the conformational distributions of IDPs respond to crowded environments is of particular current interest because IDPs have a vital role in cellular compartments and regions with very high local concentrations of proteins and RNA, such as RNA granules and nuclear pore complexes (22)(23)(24)(25).…”

mentioning

confidence: 99%

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments

Soranno

Koenig

Borgia

et al. 2014

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

233

307

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Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concentration, but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the observed behavior can be explained quantitatively if the polymeric nature of both the IDPs and the crowding molecules is taken into account explicitly. Our results suggest that excluded volume interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu. A surprisingly large number of eukaryotic proteins either contain substantial unstructured regions or are entirely unfolded under physiological conditions (1, 2). These "intrinsically disordered proteins" (IDPs) are involved in many crucial cellular processes, such as transcription, translation, and signal transduction; their functional and conformational properties are thus of great interest for a wide range of biological questions. Important advances in understanding the structures of IDPs have been made over the past decade, especially with spectroscopic techniques, e.g., NMR (3, 4), single-molecule fluorescence (5-7), and with atomistic and coarse-grained molecular simulations (8-10). In contrast with the stable folded structures we are familiar with from 50 y of structural biology, IDPs comprise highly heterogeneous and dynamic ensembles of conformations, which either lack stable tertiary structure altogether or fold only on binding their cellular targets (4). Important components of the cellular environment that affect IDPs include not only specific cellular ligands, but also pH and the concentration of salts (11,12). An additional contribution that has been difficult to investigate experimentally comes from the large number of different solutes present in a cell that do not interact with an IDP specifically, but result in an environment that is densely filled with macromolecules and metabolites (12)(13)(14). Given their lack of persistent structure, the conformations of IDPs are expected to be particularly sensitive to the effects of such molecular crowding. Indeed, first experiments indicate that some IDPs gain structure upon crowding (15), whereas others do not (16-18), but may change their dimensions (19)(20)(21). The question of how the conformational distributions of IDPs respond to crowded environments is of particular current interest because IDPs have a vital role in cellular compartments and regions with very high local concentrations of proteins and RNA, such as RNA granules and nuclear pore complexes (22)(23)(24)(25). Howeve...

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Macromolecular Crowding in the Escherichia coli Periplasm Maintains α-Synuclein Disorder

Cited by 163 publications

References 19 publications

A soluble α-synuclein construct forms a dynamic tetramer

A soluble α-synuclein construct forms a dynamic tetramer

Protein folding in confined and crowded environments

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments

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