View Single Post
  #1  
Unread 12-08-2017, 01:06 AM
nmrlearner's Avatar
nmrlearner nmrlearner is offline
Senior Member
 
Join Date: Jan 2005
Posts: 18,077
Points: 193,617, Level: 100
Points: 193,617, Level: 100 Points: 193,617, Level: 100 Points: 193,617, Level: 100
Level up: 0%, 0 Points needed
Level up: 0% Level up: 0% Level up: 0%
Activity: 50.7%
Activity: 50.7% Activity: 50.7% Activity: 50.7%
Last Achievements
Award-Showcase
NMR Credits: 0
NMR Points: 0
Downloads: 0
Uploads: 0
Default The Cu2+-nitrilotriacetic acid complex improves loading of ?-helical double histidine site for precise distance measurements by pulsed ESR

The Cu2+-nitrilotriacetic acid complex improves loading of ?-helical double histidine site for precise distance measurements by pulsed ESR

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








More...
Reply With Quote


Did you find this post helpful? Yes | No