Protein Folding Database: Measurement Report

Folding Data

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Note: A field with its data listed as "ND" denotes there is no data for that field.

[Measurement ID: 288] contact us to make corrections & updates to this data

Compare Data
compare with other measurements for this protein (1)	compare with other measurements for proteins in this SCOP Family (3)

Protein Data
Protein Name	E colicin binding Immunity Protein 9 (ImmE9) (Im9)
Oligomeric State	Monomer
Folding Mechanism	Framework
Intermediates	0
Phi Pattern	No phi value analysis
SCOP Class	Alpha \| goto SCOP
SCOP Family	Colicin E immunity proteins \| goto SCOP

Construct Data
Species	E. coli (Escherichia coli)	goto 3D view
Sequence	MELKHSISDYTEAEFLQLVTTICNADTSSEEELVKLVTHFEEMTEHPSGS DLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQG Blast PFD \| Blast NCBI NR
Length	86
Molecular Weight	9564.51Da
Fusion	None
PDB ID	1IMQ \| goto PDB
PDB Resolution	NMR
Chain	Any chain
Residues	2 - 86
SCOP ID	47352 \| goto SCOP
UniProt ID	IMM9_ECOLI \| goto UniProt
Relative Contact Order	12.09

Publication Data
Publication	Ferguson N et al (1999) Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9, J. Mol. Biol. 286, 1597-1608 goto pubmed
Data type in publication	Equilibrium & Kinetic \| view other data in this publication

Graphical Analysis Data

Contact Order Plot Data

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Note: Mutant measurements do not have their data plotted on this graph, instead the data from wildtype is marked in red.

view notes on contact order

Kinetic Data & Methods

Kinetic Unfolding Data

[Denaturant] Minimum (M)	[Denaturant] Maximum (M)	k_u (s^-1)	lnk_u (s^-1)	m_u (M^-1)	m_‡-N (kJ mol^-1 M^-1)
ND	ND	0.0124 ± 0.0017 (0M)	-4.39 ± -0.07 (0M)	ND	ND

Kinetic Folding Data

[Denaturant] Minimum (M)	[Denaturant] Maximum (M)	k_fH₂O (s^-1)	ln(k_fH₂O) (s^-1)	m_f (M^-1)	m_‡-D (kJ mol^-1 M^-1)
ND	ND	1450 ± 40	7.28 ± -0.01	ND	ND

Other Kinetic Data

[Denaturant] 50% (M)	K_D-N	m_D-N	∆G_D-NH₂O Kinetic	βT Kinetic Unfolding	βT Kinetic Folding	βT Equilibrium Unfolding	βT Equilibrium Folding
ND	ND	ND	ND	ND	0.94	ND	ND

Probe

Trp Fluorescence what is this?

Pertubation

Denaturant what is this?

Technique

Urea

Instrument

Applied Photophysics DX.17MV Sequential Mixing Stopped-flow Spectrometer

Temperature

283K

Buffer

0.05M Phosphate

Additive 1:

0.0050M DTT

Protein Concentration

0.02M

Unfolding Fit

Linear

Refolding Fit

Linear

Notes & Comments on this measurement
Measurement Notes
Protein Notes	High-resolution structure shows that the protein adopts a distorted, antiparallel four-helical structure. The small size of this protein, lack of disulphide bonds, prosthetic groups and cis-Xaa prolyl peptide bonds in the native state, makes the immunity protein ideal for folding studies. Moreover, the presence of a unique conserved tryptophan residue (W74) which is highly quenched in the native state and their all a-helical topology, make these proteins easily amenable to analysis by fuorescence and far-UV CD spectroscopy.
Construct Notes
Kinetic Notes	Kinetic unfolding and refolding measurements were performed using an Applied Photophysics SX.17MV stopped-flow fluorimeter (Leatherhead, UK). Temperature at the observation cell (measured with an in situ thermocouple) was regulated to 10(±0.1) °C using a Neslab-RTE300 circulating waterbath. Excitation was at 280 nm using a 10 nm slit width (slit widths were varied in the protein concentration dependence experiments). A bandpass filter was used to measure emitted light above 320 nm from a cuvette of 2 mm pathlength. All kinetic experiments were performed in buffered solutions containing 50 mM Na2HPO4(pH 7.0) and 2 mM DTT. Refolding experiments were performed by 1:10 dilution of unfolded protein (approximately 180 μM protein in 8 M urea, 50 mM Na2HPO4(pH 7.0), 5 mM DTT) into buffered solutions with final urea concentrations in the range 0.75–8.0 M. Unfolding experiments were performed by 1:10 dilution of native protein (180 μM initial concentration) into buffered solutions with final urea concentrations in the range 2–8.0 M. Kinetic measurements for both unfolding and refolding reactions were performed between 8 and 12 times under identical conditions. The data were then averaged and fitted to a monoexponential function using the manufacturers software. The initial and final fluorescence signals were determined from this fit to the kinetic refolding reactions.
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