Characterization of Interactions Between Proteins Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy

Crane, Jennine M.; Lilly, Angela A.; Randall, Linda L.

doi:10.1007/978-1-60327-412-8_11

“…In site-directed spin labeling, a radical containing nitroxide spin label is site specifically introduced onto cysteine residues introduced via site-directed mutagenesis. The resulting EPR spectrum of the generated paramagnetic side chain can be used to identify interaction sites within protein complexes or to detect changes in protein conformations, and the data can be interpreted in terms of relative domain movement, backbone dynamics and folding or unfolding events [26] , [27] , [28] , [29] , [30] . Moreover, pulsed EPR techniques, specifically double electron-electron resonance (DEER) experiments, allow the measurement of long-range distances and distance distributions in multi-labeled proteins [31] .…”

Section: Introductionmentioning

confidence: 99%

Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

Lorenzi

¹

,

Sylvi

²

,

Gerbaud

³

et al. 2012

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Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.

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“…S1 C and D, ΔH 0 ) and the overall breadth of the EPR spectra, especially the intensity of hyperfine splitting that arises from highly immobile populations of spin probes relative to the mobile population (Fig. 1B, dashed vs. solid arrows) (23).…”

Section: Resultsmentioning

confidence: 97%

“…We first asked whether the early intermediate forms the same or distinct interaction interface. To this end, electron paramagnetic resonance (EPR) spectroscopy was used to probe the interaction surface (23)(24)(25). Based on the cocrystal structure of the stable SRP•SR NG-domain complex, we selected residues in the vicinity of the interaction surface on SR for replacement by cysteine, which allowed site-directed spin labeling with the nitroxide probe (1-oxy-2,2,5,5-tetramethyl-3-pyrrolinyl-3-methyl) methanethiosulfonate.…”

Section: Resultsmentioning

confidence: 99%

Direct visualization reveals dynamics of a transient intermediate during protein assembly

Zhang

¹

,

Lam

²

,

Mou

³

et al. 2011

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

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Interactions between proteins underlie numerous biological functions. Theoretical work suggests that protein interactions initiate with formation of transient intermediates that subsequently relax to specific, stable complexes. However, the nature and roles of these transient intermediates have remained elusive. Here, we characterized the global structure, dynamics, and stability of a transient, on-pathway intermediate during complex assembly between the Signal Recognition Particle (SRP) and its receptor. We show that this intermediate has overlapping but distinct interaction interfaces from that of the final complex, and it is stabilized by longrange electrostatic interactions. A wide distribution of conformations is explored by the intermediate; this distribution becomes more restricted in the final complex and is further regulated by the cargo of SRP. These results suggest a funnel-shaped energy landscape for protein interactions, and they provide a framework for understanding the role of transient intermediates in protein assembly and biological regulation.EPR spectroscopy | fluorescence spectroscopy | molecular recognition | protein targeting | GTPases I nteractions between proteins are central to biology and underlie numerous molecular recognition, regulation, and signaling events (1). A challenge in our understanding of protein interactions is to reconcile their fast association kinetics required for biological function (10 6 -10 8 M −1 s −1 ) with the fact that formation of stable protein assemblies often involves extensive short-range, stereospecific interactions that are difficult to accomplish during a single diffusional encounter (2-4). This problem becomes more pronounced in protein interactions that require extensive conformational changes in the interaction partners. Much theoretical work has suggested that assembly of a protein complex initiates with the formation of a transient intermediate held together by solvent cage and long-range electrostatic attractions, followed by relative rotatory diffusions of the binding partners to search for the optimal interaction interface with shape and electrostatic complementarity (4-9). An extreme example of this concept is the "fly-casting mechanism," in which unstructured protein molecules bind targets weakly at a relatively large distance followed by folding at the target site (10-12). In general, formation of transient intermediates reduces the dimension of translational and rotational search and could significantly accelerate protein association.Despite significant progress in theoretical work, direct experimental demonstration of this model has been limited, and the structural and dynamic nature of transient intermediates during protein interactions has remained elusive. Experimental studies of transient intermediates are still at the infant stage, because these intermediates have short lifetimes and are rarely populated at equilibrium. Pioneering NMR studies have revealed the structures of rare conformational states in equilibrium with the predominant ...

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Direct Visualization Reveals Dynamics of a Transient Intermediate During Protein Assembly

Zhang¹

2011

Multistate GTPase Control Co-Translational Protein Targeting

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Interactions between proteins underlie numerous biological functions. Theoretical work suggests that protein interactions initiate with formation of transient intermediates that subsequently relax to specific, stable complexes. However, the nature and roles of these transient intermediates have remained elusive. Here, we characterized the global structure, dynamics, and stability of a transient, on-pathway intermediate during complex assembly between the Signal Recognition Particle (SRP) and its receptor. We show that this intermediate has overlapping but distinct interaction interfaces from that of the final complex, and it is stabilized by longrange electrostatic interactions. A wide distribution of conformations is explored by the intermediate; this distribution becomes more restricted in the final complex and is further regulated by the cargo of SRP. These results suggest a funnel-shaped energy landscape for protein interactions, and they provide a framework for understanding the role of transient intermediates in protein assembly and biological regulation.EPR spectroscopy | fluorescence spectroscopy | molecular recognition | protein targeting | GTPases I nteractions between proteins are central to biology and underlie numerous molecular recognition, regulation, and signaling events (1). A challenge in our understanding of protein interactions is to reconcile their fast association kinetics required for biological function (10 6 -10 8 M −1 s −1 ) with the fact that formation of stable protein assemblies often involves extensive short-range, stereospecific interactions that are difficult to accomplish during a single diffusional encounter (2-4). This problem becomes more pronounced in protein interactions that require extensive conformational changes in the interaction partners. Much theoretical work has suggested that assembly of a protein complex initiates with the formation of a transient intermediate held together by solvent cage and long-range electrostatic attractions, followed by relative rotatory diffusions of the binding partners to search for the optimal interaction interface with shape and electrostatic complementarity (4-9). An extreme example of this concept is the "fly-casting mechanism," in which unstructured protein molecules bind targets weakly at a relatively large distance followed by folding at the target site (10-12). In general, formation of transient intermediates reduces the dimension of translational and rotational search and could significantly accelerate protein association.Despite significant progress in theoretical work, direct experimental demonstration of this model has been limited, and the structural and dynamic nature of transient intermediates during protein interactions has remained elusive. Experimental studies of transient intermediates are still at the infant stage, because these intermediates have short lifetimes and are rarely populated at equilibrium. Pioneering NMR studies have revealed the structures of rare conformational states in equilibrium with the predominant ...

show abstract

Characterization of Interactions Between Proteins Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy

Cited by 4 publications

References 15 publications

Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

Direct visualization reveals dynamics of a transient intermediate during protein assembly

Direct Visualization Reveals Dynamics of a Transient Intermediate During Protein Assembly

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