TY - JOUR
T1 - Simultaneous detection of intra-and inter-molecular paramagnetic relaxation enhancements in protein complexes
AU - Olivieri, Cristina
AU - Subrahmanian, Manu Veliparambil
AU - Xia, Youlin
AU - Kim, Jonggul
AU - Porcelli, Fernando
AU - Veglia, Gianluigi
N1 - Publisher Copyright:
© Springer Science+Business Media B.V., part of Springer Nature 2018.
PY - 2018/3
Y1 - 2018/3
N2 - 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 or13C). 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 noncovalent 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.
AB - 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 or13C). 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 noncovalent 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.
KW - Intra-and inter-molecular PRE
KW - Paramagnetic relaxation enhancement
KW - Protein
KW - Protein interactions
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U2 - 10.1007/s10858-018-0165-6
DO - 10.1007/s10858-018-0165-6
M3 - Article
C2 - 29396770
AN - SCOPUS:85052367422
SN - 0925-2738
VL - 70
SP - 133
EP - 140
JO - Journal of biomolecular NMR
JF - Journal of biomolecular NMR
IS - 3
ER -