Alignment Media Preparation

Brief Description

¹H-¹⁵N residual dipolar couplings (RDCs) are easily acquired for the purpose of protein structure validation and refinement. The data can be collected on samples labeled only with ¹⁵N. Obtaining RDCs in two different alignment media greatly improve the quality of refinement and can aid in dimer structure determination; 500µl of a 0.5-1mM sample is usually sufficient for this purpose.

Data Acquisition to RDC Calculation

Refer to the linked pages for detailed descriptions of the Jmodulation Experiment RDC and HSQCTROSY RDC Measurement and RDC calculation methods.

Alignment Media Preparation

Isotropic Sample

The first sample to be observed is an isotropic sample. You may want to dilute the isotropic sample by one third (to 75% of its original concentration) to match the concentration of most aligned samples. In the case of a weak dimer, this may be important.

PEG Bicelle

Alignment of the protein sample in PEG(C₁₂E₅/hexanol) is used as a first alignment media because it produces primarily steric alignment (useful in dimer geometry predictions), the success rate is high, and it can be doped with other charged detergents to give a second alignment media .

Chemicals used for this preparation:

Sigma Aldrich 76437, Pentaethylene glycol monododecyl ether (C₁₂E₅ PEG)

Sigma Aldrich H13303, Hexanol

Sigma Aldrich 855820, Cetyltrimethylammonium bromide (CTAB)

Sigma Aldrich O4003, Sodium octyl sulfate (SOS)

The preparation procedure is as follows:

• Prepare the 16% PEG stock solution by first mixing 50µl of C₁₂E₅ (pentaethylene glycol monododecyl ether) with 200µl of buffer and 50µl of D₂O by vortexing.
• Add approximately 16µl of hexanol to the stock solution, in aliquots of 2µl with vortexing after each addition. The solution will go from clear to milky, then to translucent and viscous with lots of bubbles. Continue to add hexanol until the solution goes clear again. If it becomes milky/turbid again, you have gone past the nematic phase.
• Sample Content (for 220 µL sample):

145 µL of Protein stock solution

55 µL of 16% PEG stock solution

20 µL of D₂O

Final PEG concentration is 4.2%.

• Record the ²H splitting by running the s2pul expt with tn='lk' (for Varian instruments). The range of the splitting should be around +/-20Hz.

• PEG can be doped with either cetyltrimethyl ammonium bromide (CTAB) for positively charged proteins or sodium octyl sulphate (SOS) for negatively charged proteins. Charging the medium to be like the protein prevents association and gives higher resolution spectra. A suitable ratio of PEG:CTAB/SOS is ~30:1.

Pf1 Phage

Preparation of a Pf1 phage alignment sample is fairly straightforward. The protein sample is diluted by the alignment medium.

Chemicals used for this preparation:

ASLA Biotech P-50-P, Pf1 phage 50 mg/mL

The preparation procedure is as follows:

• Start with a protein stock 0.5-1mM and a pf1 phage stock of 50 mg/mL. Prepare a sample of 12.5 mg/mL of phage.

• Sample Content (for 220 µL sample):

145 µL of Protein stock solution

55 µL of Pf1 phage 50 mg/mL stock solution

20 µL of D₂O

Final phage concentration is 12.5 mg/mL

• Record the ²H splitting by running the s2pul expt with tn='lk' (for Varian instruments). The range of the splitting should be around +/-8-10 Hz.

Polyacrylamide Gel (Compressed and Stretched)

Chemicals used in preparing this alignment medium can are:

Bio-Rad 161-0144, 40%Acrylamide/Bis solution 19:1

Bio-Rad 161-0733, 10X TBE

Bio-Rad 161-0700, APS

Bio-Rad 161-0800, TEMED

Sigma Aldrich M7279-25G, N,N'-methylenebisacrylamide (BIS)

Sigma Aldrich 448281, (3-Acrylamidopropyl)trimethylammonium chloride solution

Sigma Aldrich 282731, 2-Acrylamido-2-methyl-1-propanesulfonic acid

Compressed Gels - Positive, Negative and Neutral

• Mix stock solutions of 40% acrylamide and N,N'-methylenebisacrylamide in a 19:1 ratio with 40% charged acrylate derivatives containing N,N'-methylenebisacrylamide as desired in a 19:1 ratio.

• Dilute the mixtures 10x with TBE buffer (0.9 M TRIS, 0.9 M borate, 0.02 M EDTA, pH 8.2) to a final 7% concentration.

• Polymerization is initiated by the addition of 0.15% ammoniumperoxide sulfate and 1% tetramethylethylenediamine (TEMED).

• To introduce negative charges, use acrylic acid or 2-acrylamido-2-methyl-1-propanesulfonic acid. Positive charge can be introduced by addition of (3-acrylamidopropyl)-trimethylammonium chloride (APTMAC) or N-(2-acryloamidoethyl) triethylammonium iodide. For neutral gels omit the charged species; for zwitterionic gels use equivalent amounts of acrylic acid and APTMAC. For 50% charged gels use 50% content of the charged specie and 50% acrylamide.

• Add 130µl of the mixture to plastic tubes with a 3.2 mm inner diameter and keep them overnight allowing polymerization to occur.

• Wash the polymerized gels extensively in deionized water (three cycles over a period of 2 days). The gels will increase in size due to electro osmotic swelling.

• Dry the gels over a 2 day period at room temperature on a teflon pan.

References

Updated by Hsiau-Wei Lee, 2011