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Compact, hydrophilic, lanthanide-binding tags for paramagnetic NMR spectroscopy
1Monash Institute of Pharmaceutical Sciences , Monash University , Parkville , VIC 3052 , Australia . Email: bim.graham@monash.edu ;
Chemical Science
|March 22, 2018
Summary
Four new lanthanide-binding tags were developed for paramagnetic NMR spectroscopy. Tags C7 and C8, with short linkers, significantly enhanced pseudocontact shifts (PCS) in protein studies.
Area of Science:
- Biophysical Chemistry
- Structural Biology
- Nuclear Magnetic Resonance (NMR) Spectroscopy
Background:
- Paramagnetic NMR spectroscopy is crucial for determining protein structures.
- Lanthanide-binding tags are essential tools for inducing pseudocontact shifts (PCS) in NMR.
- Existing tags have limitations in size, hydrophobicity, and efficiency.
Purpose of the Study:
- To design, synthesize, and evaluate novel lanthanide-binding tags for enhanced NMR studies.
- To develop tags with minimized size and hydrophobicity for improved protein conjugation.
- To assess the efficiency of new tags in inducing PCS for structural analysis.
Main Methods:
- Synthesis of four novel lanthanide-binding tags based on a chiral alcohol-scaffold.
- Conjugation of tags to proteins via a single cysteine residue using variable linkers.
- Evaluation of tag performance using ubiquitin, GB1, and HPPK protein mutants.
Main Results:
- Two novel enantiomeric tags, C7 and C8, demonstrated significantly larger Δχ-tensors compared to the C1 tag.
- The enhanced performance of C7 and C8 is attributed to their extremely short linkers, reducing lanthanide ion mobility.
- Successful demonstration of PCS induction by C7 and C8 on various protein mutants.
Conclusions:
- The developed lanthanide-binding tags, particularly C7 and C8, represent a significant advancement in paramagnetic NMR spectroscopy.
- The design incorporating short linkers effectively enhances the magnitude of induced PCS.
- These tags offer improved capabilities for structural determination of biomolecules using NMR.
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