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Pcomp Element Datasheet General
Date Deposited Peptide Name Project Name Authors Contact
29/2/2012 CC-pLI Basis Set ART,GB [email protected]
Reference Fletcher et. al., JACS (submitted); PDB: 3R4A
Summary
1. Source / design rationale Designed coiled-coil tetramer for basis set project: follows Harbury rules L@a, I@d 2. Preparation Chemical synthesis. Standard HBTU coupling on CEM liberty system. ChemMatrix rink amide resin, acetic anhydride/pyridine capping 3. Biophysical characterization CD, AUC, crystallographic studies, DLS 4. Biological characterization
Sequence details
Sequence Absorbance at 280 nm (μ-1 cm-1)
Charge at pH 7.0
Molecular Weight
G ELAAIKQ ELAAIKK ELAAIKW ELAAIKQ GAG
gabcdef gabcdef gabcdef gabcdef
xxxx +1 3375
Method of preparation
Recombinant Expression Chemical synthesis Other
Level of characterization
Proof of purity (e.g. HPLC)
Proof of identity (e.g. MS)
3D structure (NMR or XRD)
Biological Characterization
Complete?
Refer to page 2 2 6
Analysis experiments:
CD DLS AUC FT-IR NMR XRD
Page:3 Page:4 Page:5 Page: Page: Page:6
Activity:
Pcomp Element ID: BS4
CC-pIL (BS4) HPLC / MS data 2
Peptide purification by reverse-phase HPLC using a Kromatek C18HQsil column (150 by 10 mm) running a linear gradient of acetonitrile and water, each containing 0.1% TFA. A typical gradient ran from 20% to 80% acetonitrile over 30 minutes. Analytical HPLC (top) and MALDI-TOF mass spectrum for CC-pLI(bottom)
CC-pLI (BS4) CD data 3
CD spectrum (top) and temperature dependence of signal at 222nm (bottom) for CC-pLI at 50 μM concentration in PBS buffer
CC-pII-LI (BS4) Dynamic Light Scattering 4
Samples were prepared at 100 μM peptide concentration in PBS. Samples were centrifuged prior to analysis in order to remove any large particulate material. Measurements were made at 20°C using automated settings. The data was analyzed using the associated DTS Nano particle sizing software.
CC-pLI (BS4) AUC data 5
AUC fits for CC-pLI (top) and residuals (bottom). Rotor speeds were 40,000 rpm (green), 43,000 rpm (blue) and 46,000 rpm (red)
CC-pLI (BS4) Crystallography data 6
1. Crystallization conditions:
Peptide Salt Buffer pH Precipitant
CC-pLI None 0.1 M Tris 7.5 3 M HCOONa
2. Peptide used for crystallization:
Ac-GELAAIKQ ELAAIKK ELAAIKW ELAAIKQ GAG-NH2
3. Data collection and refinement statistics
PDB ID
CC-Tet
3R4A
Indexing and scaling
Space Group I 2 2 2
a, b, c (Å) 44.7, 50.9, 103.1
(°) 90, 90, 90
N° of merged
unique reflections 7490
Resolution (Å)
(outer shell)
41.03 - 2.07
(2.18 - 2.07)
Completeness (%)1 100 (99.9)
Redundancy1 6.7 (6.7)
I/σ(I)1 12.8 (2.9)
Rmerge (%)1 0.102 (0.624)
Phasing method MR
Refinement
Resolution (Å) 22.8 - 2.07
N° of reflections work
(free) 6909 (362)
Rcryst 0.209
Rfree 0.270
RMSD bond length (Å) 0.007
RMSD bond angle (°) 0.773
Coiled-coil parameters3
Radius (Å) 6.84 (0.06)
Pitch (Å) 211.3 (12)