Asla's Pf1 Nuclear Magnetic Resonance Co-Solvent

Filamentous bacteriophage for alignment RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions.

A.B. C.

Fig.1 A. Pf1 phage strain LP11-92 is isolated from wild type Pseudomonas aeruginosa and propagated in the phage free strain LA23-99. B. KBr gradient of Pf1 preparation. Phage band is marked by the yellow arrow, membrane fraction is shown by red one. C. The electron microscopy picture of the Pf1 preparation is kindly provided by Dr. Velta Ose, Biomedicine Research and Study Center, Riga, Latvia.

The phage is purified on KBr gradient and purchased in a suspension of 10mM K-phosphate buffer pH7.6, 2mM MgCl2 and 0.05% NaN3 (buffer content may be adjusted as to the need).

Description	Size	Cat#
Pf1 co-solvent, Protease free,	50 mg	P-50-P
in 10mM K-phosphate buffer pH7.6,	100 mg	P-100-P
2mM MgCl2 and 0.05% NaN3	300 mg	P-300-P
	500 mg	P-500-P

Pf1 co-solvent, RNAase and Protease free,	50 mg	P-50-RNA
in 10mM K-phosphatebuffer pH7.6,	100 mg	P-100-RNA
DEPC-treated water	300 mg	P-300-RNA
	500 mg	P-500-RNA

References on Pf1 mediated tunable alignment of proteins and nucleic acids
1. Hansen MR, Mueller L, Pardi A. (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat Struct Biol Dec;5(12):1065-74
2. Clore, GM, Starich, MR, Gronenborn, AM. (1998) Measurement of residual dipolar couplings of macromolecules aligned in the nematic phase of a colloidal suspension of rod-shaped viruses. J. Am. Chem. Soc. 120 , 10571-10572.
3. Markus MA, Gerstner RB, Draper DE, Torchia DA.(1999) Refining the overall structure and subdomain orientation of ribosomal protein S4 delta41 with dipolar couplings measured by NMR in uniaxial liquid crystalline phases. J Mol Biol Sep 17;292(2):375-87
4. Hansen MR, Hanson P, Pardi A. (2000) Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions. Methods Enzymol; 317:220-40
5. Ramirez BE, Voloshin ON, Camerini-Otero RD, Bax A.(2000) Solution structure of DinI provides insight into its mode of RecA inactivation. Protein Sci. Nov; 9 (11):2161-9
6. Zweckstetter M, Bax A.(2001) Characterization of molecular alignment in aqueous suspensions of Pf1 bacteriophage. J Biomol NMR Aug;20(4):365-77
7. Zweckstetter M. Residual dipolar couplings and orientational effects Max-Planck-Institute for Biophysical Chemistry, Göttingen http://www.mpibpc.mpg.de/abteilungen/030/zweckstetter/_lectures_files/embo2002.pdf

Tunable alignments

Quadrupolar splittings of the 2H NMR signal of D2O in the presence of different concentrations of Pf1 phage LP11-92, recorded at 25oC. The measurements are done in Karolinska Institute, Department of Medical Biochemistry and Biophysics in the group of Professor Gottfried Otting.

The relationship between LP11-92 phage concentration and delta/Hz.

Raw data of quadrupolar splitting.

BUFFER CONDITONS USED FOR ALIGNMENT OF MACROMOLECULES WITH PF1 FILAMENTOUS PHAGE

(compiled from Methods in Enzymology, 2000, Vol. 317, 220-240).

BUFFER CONDITIONS/ Normalized 2H residual coupling (Hz)
10 mM NaH2PO4, 1.5 mM EDTA, pH 8, ~17 mg/ml Pf1, 0.3 mM IRE RNA/ 8.2
40 mM NaH2PO4, 0.5 mM EDTA, pH 8, ~13 mg/ml Pf1, 1.8 mM DNA 16-me/r 6.0
10 mM NaH2PO4, 100 mM NaCI, pH 6.8, ~6 mg/ml Pf1, 1 mM DNA 10-mer/ 7.7
10 mM Tris(d11), 0.5 mM EDTA, pH 8, ~22 mg/ml Pf1, 0.9 mM IRE RNA/ 6.1
10 mM imidazole(d4), 5 mM KCl, 0.5 mM EDTA, pH 6.5, ~22 mg/ml Pf1, 1.9 mM apocalmodulin/ 7.2
10 mM succinate, 0.1 mM EDTA, pH 5.5, ~19 mg/ml Pf1/ 8.2 (7.3)
10 mM succinate, 100 mM NaCl, 0.1 mM EDTA, pH 5.5, ~19 mg/ml Pf1/ 7.8 (5.0)
10 mM succinate, 100 mM NaCl, 2.0 mM MgCl2, 0.1 mM EDTA, pH 5.5, ~20 mg/ml Pf1/ 6.9 (4.6)
10 mM Tris(d11), 0.1 mM EDTA, pH 8, ~15 mg/ml Pf1/ 8.8
10 mM Tris(d11), 0.1 mMEDTA, pH 7, ~15 mg/ml Pf1/ 5.5
10 mM Tris(d11), 0.1 mM EDTA, pH 6, ~15 mg/ml Pf1/ 4.4

The residual quadrupole coupling constants for the D2O signal were measured by 1-D 2H spectra. Because all buffer conditions did not have the same Pf1 concentration, the values given are normalized to Pf1 phage concentration of 10 mg/ml. Values in parentheses indicate that the sample was prepared by dialysis, whereas the other samples were prepared by pelleting the phage in the given buffer.