SASDEC3: Unlabeled nucleoporin NSP1 (NSP) without denaturant

Basic information

Entry

Database: SASBDB / ID: SASDEC3

Sample

Unlabeled nucleoporin NSP1 (NSP) without denaturant

• Nucleoporin NSP1 (protein), Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Function / homology

Function:

nuclear pore central transport channel / Transport of Mature mRNA derived from an Intron-Containing Transcript / Regulation of HSF1-mediated heat shock response / nuclear pore nuclear basket / tRNA export from nucleus / SUMOylation of SUMOylation proteins / SUMOylation of RNA binding proteins / structural constituent of nuclear pore / SUMOylation of chromatin organization proteins / RNA export from nucleus / nucleocytoplasmic transport / poly(A)+ mRNA export from nucleus / NLS-bearing protein import into nucleus / ribosomal large subunit export from nucleus / nuclear pore / ribosomal small subunit export from nucleus / nuclear periphery / phospholipid binding / protein import into nucleus / nuclear envelope / nuclear membrane

Domain/homology:

Nucleoporin, NSP1-like, C-terminal / Nucleoporin NSP1/NUP62 / Nsp1-like C-terminal region

Component:

Nucleoporin NSP1

• 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

Sample

• Sample: Name: Unlabeled nucleoporin NSP1 (NSP) without denaturant / Specimen concentration: 2.00-5.00
• Buffer: Name: PBS, 10 mM DTT / pH: 7.4

Entity #1249

Type: protein / Description: Nucleoporin NSP1 / Formula weight: 17.635 / Num. of mol.: 1
Source: Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
References: UniProt: P14907
Sequence:

GCNFNTPQQN KTPFSFGTAN NNSNTTNQNS STGAGAFGTG QSTFGFNNSA PNNTNNANSS ITPAFGSNNT GNTAFGNSNP TSNVFGSNNS TTNTFGSNSA GTSLFGSSSA QQTKSNGTAG GNTFGSSSLF NNSTNSNTTK PAFGGLNFGG GNNTTPSSTG NANTSNNLFG ATANANUA

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 nucleoporin NSP1 (NSP) without denaturant / Measurement date: Jun 24, 2015 / Cell temperature: 23 °C / Exposure time: 0.05 sec. / Number of frames: 20 / Unit: 1/nm /

	Min	Max
Q	0.0268	4.5256

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 987 /

	Min	Max
Q	0.0584366	2.66162
P(R) point	1	987
R	0	15

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	9.5 kDa	-
Volume	-	45.45 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	8641	87.1	8825	87.1
Radius of gyration, Rg	4.108 nm	0.06	4.1 nm	0.3

	Min	Max
D	-	15
Guinier point	13	111