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.

Figure 1. Reporters and responders of eglin c

The methyl-bearing residues that respond to the V54A mutation in eglin c are shown as a contiguous slate surface(15). The site of mutation is shown in red. Those methyl residues that were unobservable or exhibit no change are shown in gray. The “new” aromatic reporters are shown in green. The circled region is the “aromatic corner” which completes the hydrophobic core of the protein and was inaccessible in previous studies. The dashed arrows indicate residues in the back of the structure as orientated. Figures in this paper were rendered in PyMol (www.pymol.org) using a Rosetta Design(62) modeled V54A* structure based on the X-ray structure of eglin c (1CSE)(70).

Figure 2. Model-free parameters for aromatic side chains

Panel (A) Combined order parameters S²_combined (S²_combined = S² or S²_s × S²_f) are plotted for each aromatic side chain. An S² order parameter value of one corresponds to a completely rigid entity while an S² value of zero implies isotropic sampling of all possible orientations. (B) Internal correlation time constant τ_e/s, in nanoseconds, is plotted. Black bars indicate WT values and gray bars indicate V54A values. Error bars indicate standard errors, obtained from Monte Carlo simulations

Figure 3. Reduced chi-square (χ²_red =χ²/degrees of freedom (where degrees of freedom = # of data points - # of fitted parameters -1)) values from model-free fits

χ²_red values for individual fits of the model-free parameters to ¹³C relaxation data are plotted for the 13 aromatic side chains. χ²_red values were determined by comparing experimental T₁, T₂, and {¹H}-¹³C NOE data to back-calculated values. Residues W10 shows a poor fit in V54A; therefore, we place little confidence in the value fit for this residue.

Figure 4. Changes in ps-ns model-free order parameters

(A) Changes in S² (or S²_axis) upon V54A mutation. Changes shown in red are those methyl-bearing residues reported as significant by Clarkson et al. Black denotes the site of mutation. Changes highlighted in blue are aromatic residues that display significant change. Error bars indicate propagated standard error.

Figure 5. HX-determined local free energies and changes in local stabilities upon muation

Shown here are the HX-determined local stabilities for WT (Panel A), and V54A (Panel B). The horizontal lines denote globally determined stability via fluorescence-monitored GdnHCl melts. The secondary structure of eglin is displayed beneath the lower panel. Differences in local stabilities(Panel C) are shown as ΔΔG_HX (V54A-WT). Red bars indicate destabilization (increased rate of exchange) and blue bars indicate stabilization (slowed exchange).Black bars denotes the site of mutation.

Figure 6. ps-ns Dynamic network of methyl-bearing and aromatic side chains

The response of methyl-bearing and aromatic residues is shown on the structure of V54A eglin c. Level of response is denoted as a gradient from red (more flexible) to blue (more rigid). Aromatic residues are labeled with black letters. Gray sticks indicate residues with no significant response or residues with less reliable fits. Black denotes the site of the V54A mutation

Figure 7. Co-localization of significant motional changes on different timescales

(A) Residues that display a significant increase in rigidity on the ps-ns timescale are shown as a contiguous van der Waals surface in slate. V63 an inferred member of the network is shown in gray. Aromatic reporters are labeled in blue letters. Individual magenta surfaces represent amide nitrogens and carbonyl oxygens that participate in hydrogen bonds in regions where significant decreases (ΔΔG_HX <−0.5 kcal·mol⁻¹) in local stability are observed. The corresponding nitrogens are labeled in magenta letters as well. Dotted arrows indicate the labeled entity is located in the back of the structure as arranged. (B) Residues that display significant increase in flexibility on the ps-ns timescale are shown as two regions of contiguous van der Waals surfaces in red. Aromatic reporters are labeled in red letters. Individual blue surfaces represent amide nitrogens and carbonyl oxygens that participate in hydrogen bonds in regions where significant increases (ΔΔG_HX >0.5 kcal·mol⁻¹) in local stability are observed. The corresponding nitrogens are labeled in black letters.