A new alternative method to quantify residual structure in ‘unfolded’ proteins

Häckel, Marko; Konno, Takashi; Hinz, Hans‐Jürgen

doi:10.1016/s0167-4838(00)00051-0

Cited by 28 publications

(23 citation statements)

References 69 publications

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“…78 Prothymosin a is not an exception, and several other extended IDPs, such as p21, p27, a-synuclein, and phosphodiesterase g subunit, were shown to possess high resistance toward heat denaturation and aggregation, being virtually unaltered by heating to 90 C. [78][79][80][81][82][83][84][85][86][87][88] Curiously, this resistance to thermal aggregation has been used for purification of these proteins, 83,[89][90][91][92] and the indifference to heat treatment was proposed as an analytical tool for evaluation of the abundance of extended IDPs in various proteomes. 93,94 Furthermore, extended IDPs, being characterized by high percentages of charged residues and low overall hydrophobicity, do not undergo large-scale structural changes at low pH 95 and remain soluble under these extreme conditions.…”

Section: Intrinsic Disorder From the Traditional Viewpoint Of Proteinmentioning

confidence: 99%

Paradoxes and wonders of intrinsic disorder: Stability of instability

Uversky

2017

Intrinsically Disordered Proteins

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This article continues a series of short comments on the paradoxes and wonders of the protein intrinsic disorder phenomenon by introducing the "stability of instability" paradox. Intrinsically disordered proteins (IDPs) are characterized by the lack of stable 3D-structure, and, as a result, have an exceptional ability to sustain exposure to extremely harsh environmental conditions (an illustration of the "you cannot break what is already broken" principle). Extended IDPs are known to possess extreme thermal and acid stability and are able either to keep their functionality under these extreme conditions or to rapidly regain their functionality after returning to the normal conditions. Furthermore, sturdiness of intrinsic disorder and its capability to "ignore" harsh conditions provides some interesting and important advantages to its carriers, at the molecular (e.g., the cell wall-anchored accumulation-associated protein playing a crucial role in intercellular adhesion within the biofilm of Staphylococcus epidermidis), supramolecular (e.g., protein complexes, biologic liquid-liquid phase transitions, and proteinaceous membrane-less organelles), and organismal levels (e.g., the recently popularized case of the microscopic animals, tardigrades, or water bears, that use intrinsically disordered proteins to survive desiccation).

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Section: Intrinsic Disorder From the Traditional Viewpoint Of Proteinmentioning

confidence: 99%

Paradoxes and wonders of intrinsic disorder: Stability of instability

Uversky

2017

Intrinsically Disordered Proteins

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“…In the native gel, IUPs and rare heat-stable globular proteins will then be separated according to their charge/mass ratios. Combining this first dimension with an 8 M urea second step is rationalized by the usual structural indifference of IUPs to chemical denaturation by trichloroacetic acid, guanidine HCl, or urea as reported for Csd1 (9), NACP (11), ␤-casein (14), stathmin (12), and p21…”

mentioning

confidence: 95%

“…We reasoned that a straightforward technique to separate IUPs from globular proteins in a cellular extract could be established by the combination of a native gel electrophoresis of heat-treated proteins followed by a second, denaturing gel containing 8 M urea. The rationale for the first dimension is that IUPs are very often heat-stable as demonstrated for Csd1 (9), MAP2 (10), NACP (11), stathmin (12), and p21…”

mentioning

confidence: 99%

A Novel Two-dimensional Electrophoresis Technique for the Identification of Intrinsically Unstructured Proteins

Csizmók

Szollosi

Friedrich

et al. 2006

Molecular & Cellular Proteomics

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Intrinsically unstructured proteins (IUPs) lack a well defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes, but only a handful of them have so far been identified. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 M urea electrophoresis of heat-treated proteins where IUPs are expected to run into the diagonal, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. This behavior was born out by a collection of 10 known IUPs and four globular proteins. By running Escherichia coli and Saccharomyces cerevisiae extracts, several novel IUPs were also identified by mass spectrometric analysis of spots at or near the diagonal. By comparing this novel method to several other techniques, such as the PONDRா predictor, hydrophobicity-net charge plot, CD analysis, and gel filtration chromatography, it was shown to provide dependable global assessment of disorder even in dubious cases.

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“…It has been shown that the solubility and limited secondary structure of IDPs, such as p21, p27, α -synuclein, prothymosin α , and phosphodiesterase γ subunit, are virtually unaltered by heating to 90 ° C (3, 31, 43, 44, 46, 51, 68 -70, 72, 77) . Furthermore, heat treatment has been utilized for the recombinant IDP purifi cation (2,29,32,38,77) .…”

Section: Basic Principles Of the Approachmentioning

confidence: 99%

“…The rationales for this approach are considered below. Extended IDPs are often heat stable as demonstrated for Csd1 (29) , MAP2 (32) , α -synuclein (69,77) , its familial Parkinson ' s disease -related mutants (46) , β -and γ -synucleins (70) , stathmin (2) , p21 Cip1 (43) , prothymosin α (68) , C -terminal domain of caldesmon (51) , and phosphodiesterase γ (72) . Therefore, heat treatment should lead to a dissent in initial separation of the extended IDPs from globular proteins, the vast majority of which are known to aggregate and precipitate at high temperatures .…”

Section: Basic Principles Of the Approachmentioning

confidence: 99%

Large‐Scale Identification of Intrinsically Disordered Proteins

Uversky

Cortese

Tompa

et al. 2010

Instrumental Analysis of Intrinsically Disordered Proteins

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23A method to enrich cell extracts in totally unfolded proteins was investigated. A literature search revealed that 14 of 29 proteins isolated by their failure to precipitate during perchloric acid (PCA) or trichloroacetic acid (TCA) treatment were also shown experimentally to be totally disordered. A near 100,000 -fold reduction in yield was observed after 5% or 9% PCA treatment of total soluble Escherichia coli protein. Despite this huge reduction, 158 and 142 spots were observed from the 5% and the 9% treated samples, respectively, on silver -stained two -dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS -PAGE) gels loaded with 10 μ g of protein. Treatment with 1% PCA was less selective with more visible spots and a greater than threefold higher yield. A substantial yield of unprecipitated protein was ABSTRACT

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A new alternative method to quantify residual structure in ‘unfolded’ proteins

Cited by 28 publications

References 69 publications

Paradoxes and wonders of intrinsic disorder: Stability of instability

Paradoxes and wonders of intrinsic disorder: Stability of instability

A Novel Two-dimensional Electrophoresis Technique for the Identification of Intrinsically Unstructured Proteins

Large‐Scale Identification of Intrinsically Disordered Proteins

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