Exchange saturation transfer and associated NMR techniques for studies of protein interactions involving high-molecular-weight systems

Tugarinov, Vitali; Clore, G. Marius

doi:10.1007/s10858-019-00244-6

Cited by 21 publications

(28 citation statements)

References 31 publications

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“…In addition, the intrinsic transverse relaxation rates for the 2 monomeric species A ðR A 2 Þ and C ðR C 2 Þ were assumed to be equal. Because exchange between states A and B is fast on the relaxation time scale ðk ex =R B 2 >>1Þ, the values of the population of state B (p B ) and R B 2 are partially correlated and cannot be resolved easily from the 15 N CPMG relaxation dispersion and ΔR 2 data alone; decorrelation of these 2 terms is achieved by incorporation of the exchange-induced shift (δ ex ) data (53,54) that effectively places an upper bound on the population p B of the oligomeric state (species B). Excellent fits to the data are obtained for the 3-state branching model as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis

Karamanos

Tugarinov

Clore

2019

Proc. Natl. Acad. Sci. U.S.A.

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143

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J-domain chaperones are involved in the efficient handover of misfolded/partially folded proteins to Hsp70 but also function independently to protect against cell death. Due to their high flexibility, the mechanism by which they regulate the Hsp70 cycle and how specific substrate recognition is performed remains unknown. Here we focus on DNAJB6b, which has been implicated in various human diseases and represents a key player in protection against neurodegeneration and protein aggregation. Using a variant that exists mainly in a monomeric form, we report the solution structure of an Hsp40 containing not only the J and C-terminal substrate binding (CTD) domains but also the functionally important linkers. The structure reveals a highly dynamic protein in which part of the linker region masks the Hsp70 binding site. Transient interdomain interactions via regions crucial for Hsp70 binding create a closed, autoinhibited state and help retain the monomeric form of the protein. Detailed NMR analysis shows that the CTD (but not the J domain) self-associates to form an oligomer comprising ∼35 monomeric units, revealing an intricate balance between intramolecular and intermolecular interactions. The results shed light on the mechanism of autoregulation of the Hsp70 cycle via conserved parts of the linker region and reveal the mechanism of DNAJB6b oligomerization and potentially antiaggregation.

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Section: Resultsmentioning

confidence: 99%

Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis

Karamanos

Tugarinov

Clore

2019

Proc. Natl. Acad. Sci. U.S.A.

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143

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“…The experimental data are shown as circles, and the best‐fit curves to the two‐state exchange model shown in (C) are displayed as continuous lines. Note: the inclusion of the CPMG relaxation dispersion data in the global analysis of the DEST and Δ R 2 data serve to decorrelate p bound from

R \binom{bound}{2}

in the exchange regime where (

k \binom{app}{on}

+ k off )/

R \binom{bound}{2}

>1 [42,43] . The depiction of htt NT Q 7 bound (as a transparent blue helix to represent an ensemble of partially helical states) to GroEL in (D) is purely a cartoon, as we have no information on the orientation of bound htt NT Q 7 on the surface of the GroEL apical domain.…”

Section: Resultsmentioning

confidence: 99%

“…1. [42,43] The depiction of htt NT Q 7 bound (as a transparent blue helix to represent an ensemble of partially helical states) to GroEL in (D) is purely a cartoon, as we have no information on the orientation of bound htt NT Q 7 on the surface of the GroEL apical domain. The experiments were carried out with 200 μM 15 N-labeled htt NT S1).…”

Section: Figurementioning

confidence: 99%

Probing the Interaction of Huntingtin Exon‐1 Polypeptides with the Chaperonin Nanomachine GroEL

2021

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Huntington's disease arises from polyQ expansion within the exon‐1 region of huntingtin (httex1), resulting in an aggregation‐prone protein that accumulates in neuronal inclusion bodies. We investigate the interaction of various httex1 constructs with the bacterial analog (GroEL) of the human chaperonin Hsp60. Using fluorescence spectroscopy and electron and atomic force microscopy, we show that GroEL inhibits fibril formation. The binding kinetics of httex1 constructs with intact GroEL and a mini‐chaperone comprising the apical domain is characterized by relaxation‐based NMR measurements. The lifetimes of the complexes range from 100 to 400 μs with equilibrium dissociation constants (KD) of ∼1–2 mM. The binding interface is formed by the N‐terminal amphiphilic region of httex1 (which adopts a partially helical conformation) and the H and I helices of the GroEL apical domain. Sequestration of monomeric httex1 by GroEL likely increases the critical concentration required for fibrillization.

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“…A linear dependence of 15 N−Δ R 2 on protein concentration implies a direct dependence of 15 N−Δ R 2 on the pseudo-first-order association rate constant k on app ( k on app = k on [QD] free ), where k on is the second-order association rate constant (Figure C) . Also, in the exchange regime, k on app must be greater than Δ R 2 max , and the dissociation rate constant k off will be of the order of or greater than R 2 in the bound state …”

Section: Resultsmentioning

confidence: 99%

“…In such a case, based on the exchange regime, we must supplement DEST/Δ R 2 data with additional NMR experimental observables to decorrelate p B and R 2 bound . When the exchange is fast on the transverse relaxation time scale ( k ex / R 2 bound ≫ 1, where k ex = k on app + k off ), supplementing DEST/Δ R 2 data with closely related experiments such as the exchange-induced chemical shift (δ ex ) and/or CPMG-relaxation dispersion was necessary to decorrelate p bound from R 2 bound . , In our analysis, we incorporated 15 N-exchange-induced shifts ( 15 N−δ ex ) with DEST/Δ R 2 for the simultaneous analysis of the data obtained from the above experiments. The inclusion of 15 N−δ ex in the analysis led to the unambiguous determination of p bound and hence dissociation rate constant ( k off ) as well as residue-specific bound-state dynamics in the form of 15 N– R 2 bound .…”

Section: Resultsmentioning

confidence: 99%

Examining the Transient Dark State in Protein-Quantum Dot Interaction by Relaxation-Based Solution NMR

et al. 2021

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We probed the "dark" state involved in the proteinquantum dot (QD) interaction using a relaxation-based solution nuclear magnetic resonance (NMR) approach. We examined the dynamics and exchange kinetics of the ubiquitin-CdTe model system, which undergoes a fast exchange in the transverse relaxation time scale. We applied the recently developed dark-state exchange saturation transfer (DEST), lifetime line broadening (ΔR 2 ), and exchange-induced chemical shift (δ ex ) solution NMR techniques to obtain a residue-specific binding behavior of the protein on the QD surface. The variation in the estimated 15 N−R 2 bound values clearly shows the dynamic nature of bound Ub. Upon mapping the amino acid residues showing a faster relaxation rate on the electrostatic potential surface of the protein, we have determined that the interaction is preferably electrostatic, and the amino acid residues involved in binding lie on the positively charged surface of the protein. We believe that our experimental approach should provide more in-depth knowledge to engineer new hybrid protein-QD systems in the future.

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Exchange saturation transfer and associated NMR techniques for studies of protein interactions involving high-molecular-weight systems

Cited by 21 publications

References 31 publications

Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis

Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis

Probing the Interaction of Huntingtin Exon‐1 Polypeptides with the Chaperonin Nanomachine GroEL

Examining the Transient Dark State in Protein-Quantum Dot Interaction by Relaxation-Based Solution NMR

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