SASDE83: Labeled cold shock protein (CSP-Alexa488/Alexa594) with denaturant

Basic information

Entry

Database: SASBDB / ID: SASDE83

Sample

Labeled cold shock protein (CSP-Alexa488/Alexa594) with denaturant

• Cold shock-like protein (protein), CSP-488/594, Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
• Alexa Fluor™ 594 C5 Maleimide (other), Alexa594
• Alexa Fluor™ 488 C5 Maleimide (other), Alexa488

Function / homology

Function:

regulation of gene expression / nucleic acid binding / DNA binding / cytoplasm

Domain/homology:

Cold shock, CspA / Cold-shock (CSD) domain / Cold-shock (CSD) domain signature. / Cold-shock (CSD) domain profile. / Cold-shock protein, DNA-binding / 'Cold-shock' DNA-binding domain / Cold shock domain / Cold shock protein domain / Nucleic acid-binding, OB-fold

Component:

Cold shock-like protein

• Biological species: Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) (bacteria)
• 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: Labeled cold shock protein (CSP-Alexa488/Alexa594) with denaturant; Specimen concentration: 10 mg/ml / Entity id: 1245 / 1252 / 1253
• Buffer: Name: PBS, 10 mM DTT, 6 M urea, 0.3 M KCl / pH: 7.4

Entity #1245

Name: CSP-488/594 / Type: protein / Description: Cold shock-like protein / Formula weight: 6.628 / Num. of mol.: 1
Source: Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
References: UniProt: O54310
Sequence:

ACGKVKFFDS KKGYGFITKD EGGDVFVHFS AIEMEGFKTL KEGQVVEFEI QEGKKGGQUA

• Entity #1252: Name: Alexa594 / Type: other / Description: Alexa Fluor™ 594 C5 Maleimide / Formula weight: 0.886 / Num. of mol.: 1
• Entity #1253: Name: Alexa488 / Type: other / Description: Alexa Fluor™ 488 C5 Maleimide / Formula weight: 0.7 / Num. of mol.: 1

Experimental information

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

Scan

Title: Labeled cold shock protein (CSP-Alexa488/Alexa594) with denaturant (urea 6 M)
Measurement date: Jun 15, 2013 / Cell temperature: 23 °C / Exposure time: 0.05 sec. / Number of frames: 20 / Unit: 1/nm /

	Min	Max
Q	0.0768	4.5832

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 1156 /

	Min	Max
Q	0.166205	3.20473
P(R) point	1	1156
R	0	8.27

Result

Type of curve: single_conc
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	7.3 kDa	-
Volume	-	8.72 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	358.6	2.1	355.4	1.7
Radius of gyration, Rg	2.309 nm	0.02	2.2 nm	0.06

	Min	Max
D	-	8.27
Guinier point	35	196