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Calculation of z-Coordinates and Orientational Restraints Using a Metal Binding Tag

We introduce a new simple methodology allowing the measurement of 1H-15N residual dipolar couplings, dipolar shifts, and unpaired electron−amide proton distances. This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein. We have demonstrated this fusion strategy b...

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Published in:	Biochemistry (Easton) 2000-12, Vol.39 (49), p.15217-15224
Main Authors:	Gaponenko, Vadim, Dvoretsky, Alex, Walsby, Charles, Hoffman, Brian M, Rosevear, Paul R
Format:	Article
Language:	English
Subjects:	Bacterial Proteins Cobalt Electron Spin Resonance Spectroscopy - methods Gene Products, gag Magnetic Resonance Spectroscopy - methods Models, Chemical Models, Molecular Molecular Probes Nitrogen Isotopes Nuclear Magnetic Resonance, Biomolecular - methods Protein Conformation Protons Recombinant Fusion Proteins - chemistry Ribonucleases - chemistry Zinc Fingers
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cited_by	cdi_FETCH-LOGICAL-a415t-1ef2527e75a227a50ea1f2e1178279fbf5d37f3b0653b5c9e8fa2c7e3bb861533
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container_title	Biochemistry (Easton)
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creator	Gaponenko, Vadim Dvoretsky, Alex Walsby, Charles Hoffman, Brian M Rosevear, Paul R
description	We introduce a new simple methodology allowing the measurement of 1H-15N residual dipolar couplings, dipolar shifts, and unpaired electron−amide proton distances. This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein. We have demonstrated this fusion strategy by incorporating the zinc finger of the retroviral gag protein onto the C-terminus of barnase, a ribonuclease produced by Bacillus amiloliquifaciance. We show that this tag can be substituted with cobalt and manganese. Binding of cobalt to the gag zinc finger−barnase fusion protein introduced sufficient anisotropic paramagnetic susceptibility for orientation of the molecule in the magnetic field. Partial alignment permitted measurement of 1 J HN scalar couplings along with dipolar couplings. Replacement of bound cobalt with diamagnetic zinc removes the paramagnetic-induced orientation of barnase, permitting the measurement of only 1 J HN scalar couplings. Dipolar couplings, ranging from −0.9 to 0.6 Hz, were easily measured from the difference in splitting frequencies in the presence of cobalt and zinc. The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron−amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. This approach is robust and will prove powerful for global fold determination of proteins identified in genome initiatives.
doi_str_mv	10.1021/bi001381w
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The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron−amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. 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The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron−amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. 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source	American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list)
subjects	Bacterial Proteins Cobalt Electron Spin Resonance Spectroscopy - methods Gene Products, gag Magnetic Resonance Spectroscopy - methods Models, Chemical Models, Molecular Molecular Probes Nitrogen Isotopes Nuclear Magnetic Resonance, Biomolecular - methods Protein Conformation Protons Recombinant Fusion Proteins - chemistry Ribonucleases - chemistry Zinc Fingers
title	Calculation of z-Coordinates and Orientational Restraints Using a Metal Binding Tag
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