G121V dependent changes in backbone and side-chain dynamics on the ps-ns timescale.

<p>Changes in backbone <i>S</i>² (A) and τ_e (B) order parameters determined from ¹⁵N relaxation experiments. Changes in side-chain <i>S</i>²_axis (D) and τ_e,_axis (E) order parameters determined from ²H relaxation experiments. Plotted values were obtained by subtracting mutant (E^G121V:NADPH:MTX) from wild-type (E:NADPH:MTX) parameters with significant changes (>1.5σ) highlighted in red (backbone) and blue (side-chain). Significant changes are also mapped onto the structure using red spheres for backbone (C) and blue spheres for side-chain (F). Greens spheres denote residues that have significant changes in both backbone and side-chain order parameters. An area of 5 Å around the active site is highlighted in green.</p

DHFR subdomain and loop nomenclature.

<p>Structure of DHFR complexed with NADPH (purple) and methotrexate (yellow) (pdbid 1RX3). Adenosine binding domain is shown in light blue while the loops domain is shown in wheat. The Met20 (red), F–G (blue), G–H (green) loops are labeled. The site of mutation (G121V) is indicated by a cyan sphere.</p

Model-free analysis of tryptophan indole ¹⁵N relaxation within E:NADPH:MTX and E^G121V:NADPH:MTX complexes.

a<p><i>R</i>_ex values are based on model-free fits.</p

Methotrexate binding traps G121V in the closed conformation.

<p>(A) ¹H–¹⁵N HSQC spectra of E:NADPH:MTX (black) overlain with the ¹H–¹⁵N HSQC spectra of E^G121V:NADPH:MTX (red). (B) The reduced change in chemical shift is calculated using the following formula and is subsequently plotted as a function of residue number: . The changes as a result of mutation (G121V vs. WT) are shown in black and a bona fide closed–occluded change are plotted in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033252#pone.0033252-Osborne1" target="_blank">[23]</a>. The plot shows that the pattern of changes as a result of mutation can be attributed to differences in the local chemical environment and not large-scale structural change. The Met20 (residues 9–23), F–G (residues 117–131), and G–H (residues 142–149) loops are highlighted.</p

Native State Volume Fluctuations in Proteins as a Mechanism for Dynamic Allostery

Allostery enables tight regulation of protein function in the cellular environment. Although existing models of allostery are firmly rooted in the current structure–function paradigm, the mechanistic basis for allostery in the absence of structural change remains unclear. In this study, we show that a typical globular protein is able to undergo significant changes in volume under native conditions while exhibiting no additional changes in protein structure. These native state volume fluctuations were found to correlate with changes in internal motions that were previously recognized as a source of allosteric entropy. This finding offers a novel mechanistic basis for allostery in the absence of canonical structural change. The unexpected observation that function can be derived from expanded, low density protein states has broad implications for our understanding of allostery and suggests that the general concept of the native state be expanded to allow for more variable physical dimensions with looser packing