Complexity of Protein Energy Landscapes Studied by Solution NMR Relaxation Dispersion Experiments

Abstract

The millisecond timescale motions in ribonuclease A (RNase A) were studied by solution NMR CPMG and off-resonance R1ρ relaxation dispersion experiments over a wide pH and temperature range. These experiments identify three separate protein regions termed Cluster 1, Cluster 2, and R33 whose motions are governed by distinct thermodynamic parameters. Moreover each of these regions has motions with different pH dependencies. Cluster 1 shows an increase in activation enthalpy and activation entropy as the pH is lowered, whereas Cluster two exhibits the opposite behavior. In contrast the activation enthalpy and entropy of R33 show no pH dependence. Compounding the differences, Δω values for Cluster 2 are characteristic of two-site conformational exchange yet similar analysis for Cluster 1 indicates that this region of the enzyme exhibits conformational fluctuations between a major conformer and a pH-dependent average of protonated and de-protonated minor conformers.

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function

“…13). This complexity was recently studied in a series of temperature and pH dependent CPMG relaxation dispersion experiments [114]. Many residues within RNaseA showed evidence of ms motions at all temperatures (10, 15, 20, 25 °C) and pH values >6.4 (Fig.…”

“…Dynamic clusters 1 and 2 are enclosed within the blue boxes. Reprinted from [114] with permission. Copyright 2015 American Chemical Society.…”

“…Dynamic cluster 2 residues A64, C65, K66, T70, and N71 at 25 °C and pH 5.5 are shown in fuchsia, yellow, red, cyan, and green, respectively. Reprinted from [114] with permission. Copyright 2015 American Chemical Society.…”

“…In all cases conformation A is set at a reference value of 0 kcal/mol. Reprinted from [114] with permission. Copyright 2015 American Chemical Society.…”

“…Δω values determined at each of four pH values are well-described by a line of unit slope passing through the origin ( R 2 > 0.98), suggesting that the two chemical environments being sampled by cluster 2 remain unchanged over the pH range. Reprinted from [114] with permission. Copyright 2015 American Chemical Society.…”