Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements

Abstract

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

The Structure of Myristoylated Mason-Pfizer Monkey Virus Matrix Protein and the Role of Phosphatidylinositol-(4,5)-Bisphosphate in Its Membrane Binding

“…Protein preparation was described previously when we demonstrated that the C-terminal extension of MA does not affect its structure. 23 To determine the structure of myrMA, we used high-resolution multidimensional NMR spectroscopy in combination with computational methods. Myristoylation caused large changes in the 1 H/ 15 N chemical shifts when compared to the spectra of the non-myristoylated analogue; therefore, we assigned all protein resonances de novo .…”

“…We previously reported that the PPHis segment does not influence the structure of the (myr-)MA. 23 The N-terminus of the myrMA is fixed by the myristoyl group so that the first helix is located closer to the rest of the protein. Additionally, the orientation of the second helix is slightly different between the two forms.…”

“…23,26 All phosphoinositides were obtained from Echelon Biosciences Incorporated (Salt Lake City, UT, USA) and used without further purification. Samples for NMR experiments were dissolved in buffer containing 100mM phosphate (pH6), 300mM NaCl and 5mM DTT.…”

Molecular aspects of the interaction between Mason—Pfizer monkey virus matrix protein and artificial phospholipid membrane

“…Myristoylated M‐PMV matrix protein was prepared as previously described . Briefly, the gene for matrix protein, as well as eukaryotic N ‐myristoyltransferase, was inserted into E. coli DH5α strain.…”

A Mason-Pfizer Monkey Virus Gag-GFP Fusion Vector Allows Visualization of Capsid Transport in Live Cells and Demonstrates a Role for Microtubules

“…Pr78 is modified by an N-terminal myristic acid residue that is mostly buried within the folded protein [4]. Following interaction of Gag with the plasma membrane the exposure of this myristic acid moiety appears to be critical for envelopment and budding of the virus [5].…”