Journal of Biomolecular NMR

, Volume 70, Issue 3, pp 133–140 | Cite as

Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes

  • Cristina Olivieri
  • Manu Veliparambil Subrahmanian
  • Youlin Xia
  • Jonggul Kim
  • Fernando Porcelli
  • Gianluigi Veglia
Article
  • 298 Downloads

Abstract

Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein–protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes (15N or 13C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model protein–protein complexes, making this approach time consuming and expensive. Here, we show a new strategy that combines a modified pulse sequence (1HN-Γ2-CCLS) with an asymmetric labeling scheme to enable the detection of both intra- and inter-molecular PREs simultaneously using only one sample preparation. We applied this strategy to the non-covalent dimer of ubiquitin. Our method confirmed the previously identified binding interface for the transient di-ubiquitin complex, and at the same time, unveiled the internal structural dynamics rearrangements of ubiquitin upon interaction. In addition to reducing the cost of sample preparation and speed up PRE measurements, by detecting the intra-molecular PRE this new strategy will make it possible to measure and calibrate inter-molecular distances more accurately for both symmetric and asymmetric protein–protein complexes.

Keywords

Paramagnetic relaxation enhancement Protein–protein interactions Intra- and inter-molecular PRE 

Notes

Acknowledgements

This research was supported by the National Institute of Health (GM 100310 and 1S10OD021536 to G.V.). We authors would like to thank Dr. G. Li for helping in the initial setting of the experiments. The experiments were carried out at the Minnesota NMR Center.

Supplementary material

10858_2018_165_MOESM1_ESM.docx (911 kb)
Supplementary material 1 (DOCX 911 KB)

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Cristina Olivieri
    • 1
    • 3
  • Manu Veliparambil Subrahmanian
    • 1
  • Youlin Xia
    • 1
    • 4
  • Jonggul Kim
    • 1
    • 2
  • Fernando Porcelli
    • 3
  • Gianluigi Veglia
    • 1
    • 2
  1. 1.Department of Biochemistry, Molecular Biology, and BiophysicsUniversity of MinnesotaMinneapolisUSA
  2. 2.Department of ChemistryUniversity of MinnesotaMinneapolisUSA
  3. 3.DIBAF - University of Tuscia - Largo dell’UniversitàViterboItaly
  4. 4.Department of Structural BiologySt Jude Children’s Research HospitalMemphisUSA

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