Pf1 Nuclear Magnetic Resonance Cosolvent

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 customer needs).

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, G.M., Starich, M.R., Gronenborn, A.M. (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. Markus Zweckstetter. Residual dipolar couplings and orientational effects Max-Planck-Institute for Biophysical Chemistry, Göttingen mzwecks@gwdg

 

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
	Fig3. The relationship between LP11-92 phage concentration and delta/Hz
Fig.2 Raw data of quadrupolar splitting

 

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Technical data

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-mer	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-D²H 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.

 

Quality control & Technical tips

• of preparing pf1 for NMR measurements from M.Summers, Howard Hughes Medical Institute at University of Maryland, Baltimore
• Certificate of Quality, Technical tips and Appendixes in PDF format

 

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Pf1 price list 2022 (Euro, €)

	Item Id.	Amount, mg	Price, € (w/o shipping costs)
Pf1 cosolvent, protease free (in 10mM K-phosphate buffer pH7.6, 2mM MgCl2 and 0.05% NaN3)
	P-50-P	50	699.00
	P-100-P	100	1069.00
	P-300-P	300	2889.00
	P-500-P	500	4549.00
Pf1 cosolvent, RNAase and protease free (in 10mM K-phosphate buffer pH7.6, DEPC-treated water)
	P-50-RNA	50	769.00
	P-100-RNA	100	1179.00
	P-300-RNA	300	3189.00
	P-500-RNA	500	4979.00
Custom buffer
	including unlabeled isotope salts	50	+299.00
	including deuterium oxide or other labeled compounds		please enquire

 

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References

• References from Art Pardi, Marius Clore, Ad Bax, Gottfried Otting, and other World NMR Experts
• Papers that refer to pf1 magnetic resonance co-solvent from ASLA Biotech.

 

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