SASDEM2: Unlabeled Nuclear Localization Signal (NLS) from the inner nuclea...

Basic information

• Entry: Database: SASBDB / ID: SASDEM2

Sample

Unlabeled Nuclear Localization Signal (NLS) from the inner nuclear membrane protein HEH2 without denaturant

• Inner nuclear membrane protein HEH2 (protein), NLS, Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Function / homology

Function:

nuclear membrane organization / nuclear inner membrane / nuclear periphery / nuclear envelope / chromatin binding

Domain/homology:

Heh2/Src1-like / HeH/LEM domain / HeH/LEM domain / Man1/Src1, C-terminal / MAN1, winged-helix domain / Man1-Src1p-C-terminal domain / LEM/LEM-like domain superfamily

Component:

Inner nuclear membrane protein HEH2

• Biological species: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
• Citation: Journal: Proc Natl Acad Sci U S A / Year: 2017; Title: Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.; Authors: Gustavo Fuertes / Niccolò Banterle / Kiersten M Ruff / Aritra Chowdhury / Davide Mercadante / Christine Koehler / Michael Kachala / Gemma Estrada Girona / Sigrid Milles / Ankur Mishra / Patrick R Onck / Frauke Gräter / Santiago Esteban-Martín / Rohit V Pappu / Dmitri I Svergun / Edward A Lemke / ; Abstract: Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration ( ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance ( ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values and For chemically denatured proteins we obtain mutual consistency in our inferences based on and , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between and that is amplified in the absence of denaturants. Therefore, joint assessments of and combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

Contact author

• Gustavo Fuertes Vives (Institute of Biotechnology CAS v.v.i.)

Models

• Model #2262: ; Type: atomic; Comment: p-acetylphenylalanine was replaced with a cysteine.; Chi-square value: 0.632 / P-value: 0.812275; Search similar-shape structures of this assembly by Omokage search (details)
• Model #2263: ; Type: atomic / Software: (CAMPARI); Comment: p-acetylphenylalanine was replaced with a cysteine.; Chi-square value: 0.632 / P-value: 0.812275; Search similar-shape structures of this assembly by Omokage search (details)
• Model #2264: ; Type: atomic / Software: (CAMPARI); Comment: p-acetylphenylalanine was replaced with a cysteine.; Chi-square value: 0.632 / P-value: 0.812275; Search similar-shape structures of this assembly by Omokage search (details)
• Model #2265: ; Type: atomic / Software: (CAMPARI); Comment: p-acetylphenylalanine was replaced with a cysteine.; Chi-square value: 0.632 / P-value: 0.812275; Search similar-shape structures of this assembly by Omokage search (details)
• Model #2266: ; Type: atomic / Software: (CAMPARI); Comment: p-acetylphenylalanine was replaced with a cysteine.; Chi-square value: 0.632 / P-value: 0.812275; Search similar-shape structures of this assembly by Omokage search (details)
• Model #2267: ; Type: atomic / Software: (CAMPARI); Comment: p-acetylphenylalanine was replaced with a cysteine.; Chi-square value: 0.632 / P-value: 0.812275; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Unlabeled Nuclear Localization Signal (NLS) from the inner nuclear membrane protein HEH2 without denaturant; Specimen concentration: 2.00-7.50
• Buffer: Name: 25 mM HEPES, 150 mM NaCl, 10 mM DTT / pH: 7.4

Entity #1232

Name: NLS / Type: protein / Description: Inner nuclear membrane protein HEH2 / Formula weight: 5.406 / Num. of mol.: 1
Source: Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: Q03281
Sequence:

ACETNKRKRE QISTDNEAKM QIQEEKSPKK KRKKRSSKAN KPPEUA

Experimental information

• Beam: Instrument name: PETRA III EMBL P12 / City: Hamburg / 国: Germany / Type of source: X-ray synchrotron / Wavelength: 0.1 Å / Dist. spec. to detc.: 3 mm
• Detector: Name: Pilatus 2M

Scan

Title: Unlabeled Nuclear Localization Signal (NLS) from the inner nuclear membrane protein HEH2, without denaturant
Measurement date: Jan 25, 2014 / Cell temperature: 23 °C / Exposure time: 0.05 sec. / Number of frames: 20 / Unit: 1/nm /

	Min	Max
Q	0.0199	4.5094

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 820 /

	Min	Max
Q	0.122687	2.28047
P(R) point	1	820
R	0	11.64

Result

Type of curve: merged
Comments: The protein contains a penultimate non-canonical amino acid p-acetylphenylalanine (207 Da) that is represented as U (selenocysteine, 168 Da) in the amino acid sequence for the entry. Therefore, the calculated MW from sequence (MW(expected)) must be adjusted accordingly (ca. 40 Da).

	Experimental	Porod
MW	8.5 kDa	-
Volume	-	16.21 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	185.9	3	181.15	1.32
Radius of gyration, Rg	2.652 nm	0.09	2.4 nm	0.3

	Min	Max
D	-	11.64
Guinier point	40	200