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  Statistics
Version: 2.2
Last Update: 8-Jun-2007
Entries: 296
Proteins: 70
Families: 53
Species: 30
ϕ values: 230 (5 proteins)
Locations of visitors to this page
  Latest Additions
ACBP (3 WT)
FRB (6 WT)
CI2 (3 WT, 202 mutants)
HPr (1 WT, 4 mutants)
Im7 (1 WT)
En-HD (1 WT)
Im9 (2 WT)
Abp1 SH3 (1 WT)
CheW (1 WT)
U1A (1 WT)
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Fulton et al (2005) Nucleic Acids Res. 33:D279-283  |  Fulton et al (2007) Nucleic Acids Res. 35:D304-307 About PFD | Help | Glossary

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: 310] contact us to make corrections & updates to this data

Expand   Compare Data
compare with other measurements for this constructcompare with other measurements for this protein (204)compare with other measurements for proteins in this SCOP Family (204)

Expand   Protein Data
Protein Name Chymotrypsin Inhibitor 2 (CI2)   -  mutant: CI2
Oligomeric State Monomer
Folding Mechanism Nucleation-condensation
Intermediates 0
Phi Pattern CI2
SCOP Class Alpha+Beta   |   goto SCOP
SCOP Family CI-2 family of serine protease inhibitors   |   goto SCOP

Expand   Construct Data
Species Barley (Hordeum vulgare)

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Sequence MLKTEWPELVGKSVEEAKKVILQDKPEAQIIVLPVGTIVTMEYRIDRVRL
FVDKLDNIAQVPRVG

Blast PFD  |  Blast NCBI NR
Length 66
Molecular Weight 7398.78Da
Fusion None
PDB ID 2CI2   |   goto PDB  
PDB Resolution 2Å
Chain A
Residues 20 - 83
SCOP ID 54659   |   goto SCOP
UniProt ID ICI2_HORVU   |   goto UniProt
Relative Contact Order 15

Expand   Publication Data
Publication Itzhaki LS et al (1995) The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding, J. Mol. Biol. 254, 260-288  goto pubmed
Data type in publication Equilibrium & Kinetic   |   view other data in this publication

Expand   Graphical Analysis Data
Contact Order Plot Data Acyl Coenzyme A Binding Protein Hypothetical protein YjbJ from Escherichia coli E colicin binding Immunity Protein 7 (ImmE7*) E colicin binding Immunity Protein 9 (ImmE9) Lambda C1 repressor, DNA-binding domain E colicin binding Immunity Protein 9 (ImmE9) Acyl Coenzyme A Binding Protein Acyl Coenzyme A Binding Protein Internalin B, C-terminal domain Apo-azurin Chemotaxis protein CheW Fyn proto-oncogene tyrosine kinase, SH3 domain Alpha-Spectrin SH3 Domain c-src tyrosine kinase SH3 Domain Actin binding protein 1 SH3 domain Activation Domain Of Human Procarboxypeptidase A2 Chymotrypsin Inhibitor 2 Ribosomal protein L9 C-domain FK-506 binding protein Ribosomal protein L23 Acylphosphatase Ribosomal protein L9 N-domain Immunoglobulin-binding protein G Immunoglobulin light chain-binding domain of protein L c-Raf1 RBD Ribosomal protein S6 c-src tyrosine kinase SH2 domain Spliceosomal U1A protein Ubiquitin FK-506 binding protein Chymotrypsin Inhibitor 2 Chymotrypsin Inhibitor 2
generated contact order graph

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To view more data on a SCOP Class click on it's name in the legend.
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

Expand   Equilibrium Data & Methods
Equilibrium Data
[Denaturant] Minimum
(M)		 [Denaturant] 50%
(M)		 mD-N (m)
(kJ mol-1 M-1)		 mD-N <m>
(kJ mol-1 M-1)		 ∆GD-N (m)
(kJ mol-1)		 ∆GD-N <m>
(kJ mol-1)
ND 4 ± 0.01 7.94 ± 0.13 8.01 ± 0.05 31.77 ± 0.51 32.04 ± 0.2
Probe Trp Fluorescence  what is this?
Pertubation Denaturant  what is this?
Technique GdnHCl
Instrument Perkin Elmer LS5B Luminescence Spectrometer
Temperature 298K
pH 6.25
Buffer 0.05M Mes
Protein Concentration ND

Expand   Kinetic Data & Methods
Kinetic Unfolding Data
[Denaturant] Minimum
(M)		 [Denaturant] Maximum
(M)		 ku
(s-1)		 lnku
(s-1)		 mu
(M-1)		 m‡-N
(kJ mol-1 M-1)
ND ND 0.00012 ± ND (0M) -9.04 ± 0.07 (0M) 1.31 ± 0.01 3.25 ± 0.02
Kinetic Folding Data
[Denaturant] Minimum
(M)		 [Denaturant] Maximum
(M)		 kfH2O
(s-1)		 ln(kfH2O)
(s-1)		 mf
(M-1)		 m‡-D
(kJ mol-1 M-1)
ND ND 56.26 ± 0.05 4.03 ± 0.04 -1.82 ± 0.12 4.51 ± -0.3
Other Kinetic Data
[Denaturant] 50%
(M)		 KD-N mD-N ∆GD-NH2O Kinetic βT Kinetic Unfolding βT Kinetic Folding βT Equilibrium Unfolding βT Equilibrium Folding
ND ND 7.76 ± 0.3 ND 0.58 ± 0.02 0.58 ± 0.02 0.59 ± 0.01 0.57 ± 0.04
Probe Trp Fluorescence  what is this?
Pertubation Denaturant  what is this?
Technique GdnHCl
Instrument Perkin-Elmer MPF-44B Fluorescence Spectrophotometer
Temperature 298K
pH 6.25
Buffer 0.05M Mes
Protein Concentration ND
Unfolding Fit Linear
Refolding Fit Linear

Expand   Mutant Data
Mutation Name CI2
Mutation Type
Contacts
Location
Secondary Structure
Local Interactions The core consists of 12 residues with hydrophobic side-chains; Trp5; Leu8; Ala16; Val19; Ile20; Ala27; Ile29; Val47; Leu49; Val51; Ile57 and Pro61
Asa ND
Buried Asa ND
PDB

Expand   Notes & Comments on this measurement
Measurement Notes FF and FU are related by the expression FF + FU = 1; so that FF = 1 - FU. FFH2O from the folding experiments is the most accurate since data measured in H2O are directly compared. FU4M is less accurate since unfolding data are extrapolated to 4 M GdmCl. FU H2O is the least accurate since there is a long extrapolation of unfolding data at high [GdmCl] to 0 M.
Protein Notes The folding of CI2 is simplified by the lack of disulphide bridges and cis-peptidyl-prolyl bonds in the native state. The protein consists of a single domain (or module) that does not have readily discernible substructures that make interactions primarily within themselves. Previous studies have established that the folding and unfolding of wild-type CI2 and a range of mutants conform to a single two-state model under both equilibrium and non-equilibrium conditions (Jackson & Fersht, 1991, Jackson et al., 1993). In particular, the ratio of rate constants for unfolding and refolding give the correct equilibrium constant for unfolding (Jackson & Fersht, 1991a; Jackson et al., 1993b).
Construct Notes
Mutant Notes
Equilibrium Notes All chemicals are as described in previous papers (Jackson & Fersht, 1991a; Jackson et al., 1993a) . Mutagenesis, expression and purification of the wild-type and mutant proteins is as described previously (Jackson et al., 1993a) . The buffer used in the equilibrium and kinetic folding experiments was 2-(N-morpholino)ethanesulfonic acid (MES) purchased from Sigma. A 1 M stock solution of MES pH 6.25 contained 415 mM of the free acid and 585 mM of the sodium salt. 16 novel mutant proteins are presented in this paper: Lys→Met2, Lys→Ala2, Glu→Ala7, LysA→Αla2/Glu→Ala7, Lys→Ala2/Asp→Ala23, Ala→Gly16, Asp→Ala23, Thr→Ala36, Tyr→Ala42, Tyr→Gly42, Arg→Ala43, Asp→Ala45, Arg→Ala43/Asp→Ala45, Asp→Ala52, Asp→Asn52, Pro→Ala61. The intrinsic fluorescence of CI2 increases on denaturation, allowing unfolding and refolding to be monitored by fluorescence spectroscopy. The maximal change in fluorescence upon denaturation is obtained with an excitation wavelength of 280 nm and an emission wavelength of 356 nm. All experiments were performed at 25 ˚C. A Perkin Elmer LS5B luminescence spectrometer was used for the GdmCl equilibrium denaturation experiments, with a bandpass of 10 nm. The GdmCl solutions were prepared using a Hamilton Microlab M robot by aliquoting appropriate volumes of a solution of 7.5 M GdmCl containing 50 mM MES, pH 6.25, and a solution of 50 mM MES, pH 6.25. For each data point in the denaturation experiment, 100 μL of CI2 stock solution in 50 mM MES, pH 6.25, were diluted into 800 μL of the appropriate denaturant concentration, using a SMI positive displacement pipetter. Final concentrations were 2.5 μM CI2 and 50 mM MES, pH 6.25; final concentrations of GdmCl were 0 M to 5.5 M, in 0.1 M or 0.2 M increments. The protein-denaturant solutions were equilibrated at 25 ˚C for approximately 1 h before measuring their fluorescence.
Kinetic Notes All chemicals are as described in previous papers (Jackson & Fersht, 1991a; Jackson et al., 1993a) . Mutagenesis, expression and purification of the wild-type and mutant proteins is as described previously (Jackson et al., 1993a) . The buffer used in the equilibrium and kinetic folding experiments was 2-(N-morpholino)ethanesulfonic acid (MES) purchased from Sigma. A 1 M stock solution of MES pH 6.25 contained 415 mM of the free acid and 585 mM of the sodium salt. 16 novel mutant proteins are presented in this paper: Lys→Met2, Lys→Ala2, Glu→Ala7, LysA→Αla2/Glu→Ala7, Lys→Ala2/Asp→Ala23, Ala→Gly16, Asp→Ala23, Thr→Ala36, Tyr→Ala42, Tyr→Gly42, Arg→Ala43, Asp→Ala45, Arg→Ala43/Asp→Ala45, Asp→Ala52, Asp→Asn52, Pro→Ala61. Kinetic unfolding experiments Reactions were followed with a Perkin-Elmer MPF-44B fluorescence spectrophotometer equipped with a rapid mixing head. The mixing device contained a T-jet mixing chamber followed by a 30 ms delay loop ensuring complete mixing of the solutions before observation. A Hellma flow-through cell (10 mm x 3 mm x 3 mm) was used. The solutions were driven through the mixing chamber manually from two Hamilton syringes resulting in a mixing ratio of 1:10. The observation cell and reservoir syringes were thermostatted separately using two water baths and the temperature of each was monitored using an Edale Instrument Thermometer and maintained to ± 0.1 ˚C. Data were acquired with a Tandon Target Microcomputer, a DT2801 Data Translation board and the Bio-kine data acquisition software package (Bio-logic), and analysed using the programme Kaleidagraph (Abelbeck Software). Unfolding was initiated by rapidly diluting 1 volume of the protein solution (approximately 20 µM) in 50 mM MES, pH 6.25, with 10 volumes of concentrated GdmCl solution (containing 50 mM MES, pH 6.25). This resulted in final GdmCl concentrations of between 3 and 7 M. The lowest denaturant concentration was chosen to result in at least 98 % unfolded protein. An excitation wavelength of 280 nm and an emission wavelength of 356 nm were used. Excitation and emission slit bandwidths of 10 nm were used. Kinetic refolding experiments Refolding reactions above 0.5 M GdmCl were monitored using the same apparatus set-up as for unfolding experiments. Refolding was initiated by rapid 1:11 dilution of unfolded protein (approximately 20 µM) in 6.5 M GdmCl and 50 mM MES pH 6.25 into different concentrations of GdmCl solution in 50 mM MES pH 6.25. For lower final concentrations of GdmCl, an Applied Photophysics Stopped-Flow Spectrophotometer Model SF 17MV was used. The temperature of the cell and reservoir syringes was maintained by thermostatting with a Grant LTD6 water bath. Temperatures were maintained to ± 0.1 ˚C based on an internal temperature probe in the stopped flow apparatus which had previously been calibrated against an Edale Instrument Thermometer. Protein was initially denatured by addition of HCl to a concentration of 20 mM (pH 1.7). Refolding was initiated by rapid mixing of 1 volume of the protein with 1 volume of 100 mM MES, pH 6.65 (consisting of 21.5 mM of the free acid and 78.5 mM of the sodium salt). The final pH of the solution was pH 6.25, 50 mM MES, and the final protein concentration was approximately 10 µM. Refolding was performed in the absence and in the presence of low concentrations (up to 0.6 M, with 0.1 M increments) of GdmCl. An excitation wavelength of 280 nm was used, and slit bandwith of 2 nm. A glass cut-off filter was used to allow emission above 315 nm to be monitored. Refolding rates obtained from the two different sets of refolding experiments were identical within error when unfolded protein was refolded into GdmCl concentrations that could be monitored by both the Perkin-Elmer MPF-44B fluorescence spectrophotometer and the Applied Photophysics Stopped-Flow Spectrophotometer.
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