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 ps-ns 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 ps-ns timescale 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 K. 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 τ e values that are approximately one nanosecond. 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 timescale was probed in response to the V54A perturbation. Surprisingly, regional stabilization (slowed exchange) 10-12 angstroms from the site of mutation was observed despite a global destabilization of 1.5 kcal·mol −1 . Furthermore, this unlikely pocket of stabilized residues co-localizes with increases in aromatic flexibility on the faster timescale. Because the converse is also true (destabilized residues co-localize with rigidification on the fast timescale), a plausible entropy-driven mechanism is discussed for relating co-localization of opposing dynamic trends on vastly different timescales. KeywordsNMR; dynamics; relaxation; aromatic; hydrogen exchange; eglin c; V54AMotional flexibility, or dynamics, is essential for protein stability and function (2-6). Recently, native-state protein dynamics have been thought to be crucial for mediating allosteric signaling, or stated more generally, long-range communication (7)(8)(9)(10)(11)(12)(13)(14). One of the major challenges is the experimental characterization of dynamic processes relevant to intra- † This research was supported by NIH Grant GM066009 * To whom correspondence should be addressed: University of North Carolina, School of Pharmacy, Beard Hall, CB# 7360, Chapel Hill, NC 27599-7360. drewlee@unc.edu. Phone: (919) 843-5150. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 23. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript molecular signal transduction. Towards this goal, NMR spectroscopy is uniquely suited to non-invasively characterize both structure and dynamics in a site-specific manner. Mapping flexibility by NMR is therefore an important application for understanding signaling in prote...

Multi-Timescale Dynamics Study of FKBP12 Along the Rapamycin–mTOR Binding Coordinate

“…Nanosecond timescale motions were also observed in eglin c, the only other system for which these data have been collected with high sensitivity. 40 Taken together, these data and those for eglin c suggest that nanosecond motions may be common to aromatic side chains. Given the bulky nature of aromatic side chains and the fact that 1 H-13 C bond vector order parameters report on the displacement of the entire planar ring, 40 it is reasonable to predict that flexibility would be dependent on the degree of side-chain packing.…”

“…40 Taken together, these data and those for eglin c suggest that nanosecond motions may be common to aromatic side chains. Given the bulky nature of aromatic side chains and the fact that 1 H-13 C bond vector order parameters report on the displacement of the entire planar ring, 40 it is reasonable to predict that flexibility would be dependent on the degree of side-chain packing. To test this, we used NACCESS 45 to calculate the accessible surface area (ASA) of amino acids within free apo-FKBP [Protein Data Bank (PDB) ID 1D6O], and no correlation between aromatic order parameters and ASA was observed ( Fig.…”

“…Specifically, the δ carbons of Phe and Tyr, the δ2 and ɛ1 carbons of His, and the δ1 and ɛ3 carbons of Trp are labeled with 13 C. In order to measure the ps-ns dynamics of aromatic bond vectors, we modified the canonical suite of 15 N relaxation experiments to measure 13 C R 1 and R 2 rates and the { 1 H}-13 C heteronuclear NOE. 40 Previous studies on the protease inhibitor eglin c demonstrated that these data could be robustly fitted to model-free forms of the spectral density function. 40 The same strategy is applied here to free FKBP12 and rapamycin-bound FKBP12.…”

“…40 Previous studies on the protease inhibitor eglin c demonstrated that these data could be robustly fitted to model-free forms of the spectral density function. 40 The same strategy is applied here to free FKBP12 and rapamycin-bound FKBP12. It should be noted that the two δ carbons within Phe and Tyr are labeled with 13 C, yet single 1 H-13 C resonances are observed in every case.…”

Prepaying the entropic cost for allosteric regulation in KIX

“…Thus, our analyses show that effectorbound KIX lowers the entropic cost for binding the complementary peptide at the second site, and this is achieved by stabilizing the KIX structure. The observed reduction in entropy due to effector binding explains the decrease in k off and the small increase in k on and this notion of "prepaying the entropic cost" in allostery has been found to play a role in the tryptophan RNA binding attenuation protein (TRAP) (63,64) and the catabolite activator protein (CAP) (5,65,66). Describing allostery within a thermodynamic framework (3-6) allows us to organize the information in a quantitative manner, and it enables comparisons between entropically driven and/or enthalpically driven allosteric mechanisms.…”