Theory, Practice, and Applications of Paramagnetic Relaxation Enhancement for the Characterization of Transient Low-Population States of Biological Macromolecules and Their Complexes

Clore, G. Marius; Iwahara, Junji

doi:10.1021/cr900033p

Cited by 691 publications

(874 citation statements)

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“…In recent years, significant progress has been made in understanding protein-protein interactions at the molecular level by both experimentation and simulation (Clore and Iwahara 2009). However, most studies have been performed in dilute solutions-in vitro or in silico-which are not representative of in vivo conditions.…”

Section: Introductionmentioning

confidence: 99%

Protein–protein interactions in a crowded environment

2013

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Protein-protein interactions are important in many essential biological functions, such as transcription, translation, and signal transduction. Much progress has been made in understanding protein-protein association in dilute solution via experimentation and simulation. Cells, however, contain various macromolecules, such as DNA, RNA, proteins, among many others, and a myriad of non-specific interactions (usually weak) are present between these cellular constituents. In this review article, we describe the important developments in recent years that have furthered our understanding and even allowed prediction of the consequences of macromolecular crowding on protein-protein interactions. We outline the development of our crowding theory that can predict the change in binding free energy due to crowding quantitatively for both repulsive and attractive protein-crowder interactions. One of the most important findings from our recent work is that weak attractive interactions between crowders and proteins can actually destabilize protein complex formation as opposed to the commonly assumed stabilizing effect predicted based on traditional crowding theories that only account for the entropicexcluded volume effects. We also discuss the implications of macromolecular crowding on the population of encounter versus specific native complex.

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Section: Introductionmentioning

confidence: 99%

Protein–protein interactions in a crowded environment

2013

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“…Recently, however, the application of NMR paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to the presence of lowly populated states in the fast exchange regime (12, 13), has offered new insights into the physicochemical and structural nature of transient encounter complexes in protein-DNA (14) and protein-protein (15-20) association. The mechanism, however, whereby the interacting molecules in the encounter complex ensemble find their final stereospecific structure, remains poorly understood.The underlying principle behind this application of the PRE is as follows (13,14). The transverse PRE rate (Γ 2 ) is dependent on the sixth root of the distance between the unpaired electron of the paramagnetic center and the observed proton, and the Γ 2 rate at short distances is very large owing to the large magnetic moment of the unpaired electron (21).…”

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confidence: 99%

“…The underlying principle behind this application of the PRE is as follows (13,14). The transverse PRE rate (Γ 2 ) is dependent on the sixth root of the distance between the unpaired electron of the paramagnetic center and the observed proton, and the Γ 2 rate at short distances is very large owing to the large magnetic moment of the unpaired electron (21).…”

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confidence: 99%

“…Despite the importance of encounter complex ensembles in protein-protein association, little is known of their structures and configurations because their populations are low, their lifetimes are short, and they are difficult to trap, rendering them essentially invisible to conventional structural and biophysical methods. Recently, however, the application of NMR paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to the presence of lowly populated states in the fast exchange regime (12,13), has offered new insights into the physicochemical and structural nature of transient encounter complexes in protein-DNA (14) and protein-protein (15)(16)(17)(18)(19)(20) association. The mechanism, however, whereby the interacting molecules in the encounter complex ensemble find their final stereospecific structure, remains poorly understood.…”

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confidence: 99%

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Mechanistic details of a protein–protein association pathway revealed by paramagnetic relaxation enhancement titration measurements

Fawzi

Doucleff

Suh

et al. 2010

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

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109

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Protein-protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermolecular paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to lowly populated states in the fast exchange regime, to characterize the mechanistic details of the transient encounter complex interactions between the N-terminal domain of Enzyme I (EIN) and the histidine-containing phosphocarrier protein (HPr), two major bacterial signaling proteins. Experiments were conducted at an ionic strength of 150 mM NaCl to eliminate any spurious nonspecific associations not relevant under physiological conditions. By monitoring the dependence of the intermolecular transverse PRE (Γ 2 ) rates measured on 15 N-labeled EIN on the concentration of paramagnetically labeled HPr, two distinct types of encounter complex configurations along the association pathway are identified and dissected. The first class, which is in equilibrium with and sterically occluded by the specific complex, probably involves rigid body rotations and small translations near or at the active site. In contrast, the second class of encounter complex configurations can coexist with the specific complex to form a ternary complex ensemble, which may help EIN compete with other HPr binding partners in vivo by increasing the effective local concentration of HPr even when the active site of EIN is occupied.enzyme I-histidine containing phosphocarrier protein complex | encounter complex | lowly populated states | NMR | phosphotransferase system S pecific protein-protein interactions underlie virtually every process in the cell. In general, specific protein-protein recognition proceeds via a two-step process (1-3): weak association via diffusion-controlled intermolecular collisions results in the formation of an ensemble of short-lived, encounter complexes located in multiple local free energy minima of a two-dimensional funnel-like energy landscape on the protein surface (4); subsequent rearrangement along the energy landscape, involving translations and rotations of the two partner proteins, permits the global free energy minimum to be located, resulting in the formation of a well-defined specific complex stabilized by a defined set of electrostatic and van der Waals interactions. From a functional perspective, encounter complex ensembles are thought to play a critical role in fine tuning reaction fluxes inside the cell (5), enhancing association on-rates by increasing the interaction crosssection and reducing the conformational search space on the path to the specific complex (6-11). Despite the importance of encounter complex ensembles in...

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“…PRE can detect minor conformation of a protein when the exchange kinetics between major and minor conformations is rapid (faster than ~100 μs) and the nucleus in the minor conformation is much closer to the PRE reagent than that in the major conformation [68]. We therefore applied a solvent PRE probe, TEMPOL (4-hydroxy-2,2,6,6, tetramethylpiperidin-1-oxyl), into the hCD3ε TMCD -POPG bicelle sample ( Figure 5A).…”

Section: Heterogeneous Dynamics Of the Different Regions Of Cd3ε Cytomentioning

confidence: 99%

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor

Guo

Yan

et al. 2017

Cell Res

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T-cell receptor-CD3 complex (TCR) is a versatile signaling machine that can initiate antigen-specific immune responses based on various biochemical changes of CD3 cytoplasmic domains, but the underlying structural basis remains elusive. Here we developed biophysical approaches to study the conformational dynamics of CD3ε cytoplasmic domain (CD3ε CD ). At the single-molecule level, we found that CD3ε CD could have multiple conformational states with different openness of three functional motifs, i.e., ITAM, BRS and PRS. These conformations were generated because different regions of CD3ε CD had heterogeneous lipid-binding properties and therefore had heterogeneous dynamics. Live-cell imaging experiments demonstrated that different antigen stimulations could stabilize CD3ε CD at different conformations. Lipid-dependent conformational dynamics thus provide structural basis for the versatile signaling property of TCR.

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Theory, Practice, and Applications of Paramagnetic Relaxation Enhancement for the Characterization of Transient Low-Population States of Biological Macromolecules and Their Complexes

Cited by 691 publications

References 244 publications

Protein–protein interactions in a crowded environment

Protein–protein interactions in a crowded environment

Mechanistic details of a protein–protein association pathway revealed by paramagnetic relaxation enhancement titration measurements

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor

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