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Monitoring aromatic picosecond to nanosecond dynamics in proteins via 13C relaxation: expanding perturbation mapping of the rigidifying core mutation, V54A, in eglin c.

ABSTRACT: Long-range effects, such as allostery, have evolved in proteins as a means of regulating function via communication between distal sites. An NMR-based perturbation mapping approach was used to more completely probe the dynamic response of the core mutation V54A in the protein eglin c by monitoring changes in picosecond to nanosecond aromatic side-chain dynamics and H/D exchange stabilities. Previous side-chain dynamics studies on this mutant were limited to methyl-bearing residues, most of which were found to rigidify on the picosecond to nanosecond time scale in the form of a contiguous "network". Here, high precision (13)C relaxation data from 13 aromatic side chains were acquired by applying canonical relaxation experiments to a newly developed carbon labeling scheme [Teilum et al. (2006) J. Am. Chem. Soc. 128, 2506-2507]. The fitting of model-free parameters yielded S (2) variability which is intermediate with respect to backbone and methyl-bearing side-chain variability and tau e values that are approximately 1 ns. Inclusion of the aromatic dynamic response results in an expanded network of dynamically coupled residues, with some aromatics showing increases in flexibility, which partially offsets the rigidification in methyl side chains. Using amide hydrogen exchange, dynamic propagation on a slower time scale was probed in response to the V54A perturbation. Surprisingly, regional stabilization (slowed exchange) 10-12 A from the site of mutation was observed despite a global destabilization of 1.5 kcal x mol (-1). Furthermore, this unlikely pocket of stabilized residues colocalizes with increases in aromatic flexibility on the faster time scale. Because the converse is also true (destabilized residues colocalize with rigidification on the fast time scale), a plausible entropy-driven mechanism is discussed for relating colocalization of opposing dynamic trends on vastly different time scales.

SUBMITTER: Boyer JA

PROVIDER: S-EPMC3062916 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Monitoring aromatic picosecond to nanosecond dynamics in proteins via 13C relaxation: expanding perturbation mapping of the rigidifying core mutation, V54A, in eglin c.

Boyer Joshua A JA Lee Andrew L AL

Biochemistry 20080405 17

Long-range effects, such as allostery, have evolved in proteins as a means of regulating function via communication between distal sites. An NMR-based perturbation mapping approach was used to more completely probe the dynamic response of the core mutation V54A in the protein eglin c by monitoring changes in picosecond to nanosecond aromatic side-chain dynamics and H/D exchange stabilities. Previous side-chain dynamics studies on this mutant were limited to methyl-bearing residues, most of which ...[more]

PMID: 18393447

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Project description:The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly (13)C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular (13)C-(13)C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D (1)H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for (13)C T1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ~200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks, which are often difficult to resolve in the parent 2D spectra.

Project description:The formation of a complex between Rac1 and the cytoplasmic domain of plexin-B1 is one of the first documented cases of a direct interaction between a small guanosine 5'-triphosphatase (GTPase) and a transmembrane receptor. Structural studies have begun to elucidate the role of this interaction for the signal transduction mechanism of plexins. Mapping of the Rac1 GTPase surface that contacts the Rho GTPase binding domain of plexin-B1 by solution NMR spectroscopy confirms the plexin domain as a GTPase effector protein. Regions neighboring the GTPase switch I and II regions are also involved in the interaction and there is considerable interest to examine the changes in protein dynamics that take place upon complex formation. Here we present main-chain nitrogen-15 relaxation measurements for the unbound proteins as well as for the Rho GTPase binding domain and Rac1 proteins in their complexed state. Derived order parameters, S2, show that considerable motions are maintained in the bound state of plexin. In fact, some of the changes in S2 on binding appear compensatory, exhibiting decreased as well as increased dynamics. Fluctuations in Rac1, already a largely rigid protein on the picosecond-nanosecond timescale, are overall diminished, but isomerization dynamics in the switch I and II regions of the GTPase are retained in the complex and appear to be propagated to the bound plexin domain. Remarkably, fluctuations in the GTPase are attenuated at sites, including helices alpha6 (the Rho-specific insert helix), alpha7 and alpha8, that are spatially distant from the interaction region with plexin. This effect of binding on long-range dynamics appears to be communicated by hinge sites and by subtle conformational changes in the protein. Similar to recent studies on other systems, we suggest that dynamical protein features are affected by allosteric mechanisms. Altered protein fluctuations are likely to prime the Rho GTPase-plexin complex for interactions with additional binding partners.

Dataset Information

Monitoring aromatic picosecond to nanosecond dynamics in proteins via 13C relaxation: expanding perturbation mapping of the rigidifying core mutation, V54A, in eglin c.

Publications

Monitoring aromatic picosecond to nanosecond dynamics in proteins via 13C relaxation: expanding perturbation mapping of the rigidifying core mutation, V54A, in eglin c.

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