Pcomp Element ID: BS4

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)