Publication date: Available online 7 December 2017 Source:Journal of Magnetic Resonance
Author(s): Shreya Ghosh, Matthew J. Lawless, Gordon S. Rule, Sunil Saxena
Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu2+. This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu2+-based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu2+ ion, has till, now been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards ?-helices and incomplete Cu2+-IDA complexation. Herein, we introduce an alternative method of dHis-Cu2+ loading using the nitrilotriacetic acid (NTA)-Cu2+ complex. We find that the Cu2+-NTA complex shows a four-fold increase in selectivity toward ?-helical dHis sites. Furthermore, we show that 100% Cu2+-NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu2+ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an ?-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand ?-sheet. Finally, the DEER signal is increased twofold within two ?-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced ?-helical selectivity of Cu2+-NTA. Graphical abstract
[NMR paper] The determination of pair distance distribution by double electron-electron resonance: Regularization by the length of distance discretization with Monte Carlo calculations
The determination of pair distance distribution by double electron-electron resonance: Regularization by the length of distance discretization with Monte Carlo calculations
Publication date: Available online 2 June 2016
Source:Journal of Magnetic Resonance</br>
Author(s): Sergei A. Dzuba</br>
Pulsed double electron-electron resonance technique (DEER, or PELDOR) is applied to study conformations and aggregation of peptides, proteins, nucleic acids, and other macromolecules. For a pair of spin labels, experimental data allows for determination of their distance...
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[NMR paper] A simple double quantum coherence ESR sequence that minimizes nuclear modulations in Cu2+-ion based distance measurements
A simple double quantum coherence ESR sequence that minimizes nuclear modulations in Cu2+-ion based distance measurements
Publication date: Available online 29 May 2015
Source:Journal of Magnetic Resonance</br>
Author(s): Sharon Ruthstein , Ming Ji , Byong-kyu Shin , Sunil Saxena</br>
Double quantum coherence (DQC) ESR is a sensitive method to measure magnetic dipolar interactions between spin labels. However, the DQC experiment on Cu2+ centers presents a challenge at X-band. The Cu2+ centers are usually coordinated to histidine residues in proteins. The...
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Probing Microscopic Architecture of Opaque Heterogeneous Systems Using Double-Pulsed-Field-Gradient NMR
Probing Microscopic Architecture of Opaque Heterogeneous Systems Using Double-Pulsed-Field-Gradient NMR
Noam Shemesh, Tal Adiri and Yoram Cohen
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja200303h/aop/images/medium/ja-2011-00303h_0005.gif
Journal of the American Chemical Society
DOI: 10.1021/ja200303h
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/W-dJo6Q8sIM
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[NMR paper] The closed state of a H+ channel helical bundle combining precise orientational and d
The closed state of a H+ channel helical bundle combining precise orientational and distance restraints from solid state NMR.
Related Articles The closed state of a H+ channel helical bundle combining precise orientational and distance restraints from solid state NMR.
Biochemistry. 2002 Nov 5;41(44):13170-7
Authors: Nishimura K, Kim S, Zhang L, Cross TA
An interhelical distance has been precisely measured by REDOR solid-state NMR spectroscopy in the transmembrane tetrameric bundle of M2-TMP, from the M2 proton channel of the influenza A viral...
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11-24-2010 08:58 PM
[NMR paper] Solid-state REDOR NMR distance measurements at the ligand site of a bacterial chemota
Solid-state REDOR NMR distance measurements at the ligand site of a bacterial chemotaxis membrane receptor.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles Solid-state REDOR NMR distance measurements at the ligand site of a bacterial chemotaxis membrane receptor.
Biochemistry. 1997 Feb 18;36(7):1699-703
Authors: Wang J, Balazs YS, Thompson LK
The Escherichia coli serine receptor senses serine levels in the environment and transmits this information across the bacterial inner membrane to...
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08-22-2010 03:31 PM
[NMR paper] Solid-state REDOR NMR distance measurements at the ligand site of a bacterial chemota
Solid-state REDOR NMR distance measurements at the ligand site of a bacterial chemotaxis membrane receptor.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles Solid-state REDOR NMR distance measurements at the ligand site of a bacterial chemotaxis membrane receptor.
Biochemistry. 1997 Feb 18;36(7):1699-703
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The Escherichia coli serine receptor senses serine levels in the environment and transmits this information across the bacterial inner membrane to...
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08-22-2010 03:03 PM
[NMR paper] NMR studies of the metal-loading kinetics and acid-base chemistry of DOTA and butylam
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Bioconjug Chem. 1999 May-Jun;10(3):454-63
Authors: Keire DA, Kobayashi M
The conjugation of a chelating agent to a protein via a covalent linkage has been previously reported to change the metal-binding characteristics of the chelator. A fundamental...
The Cu2+-nitrilotriacetic acid complex improves loading of ?-helical double histidine site for precise distance measurements by pulsed ESR
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