The Energetics of a Three‐State Protein Folding System Probed by High‐Pressure Relaxation Dispersion NMR Spectroscopy

Abstract

The energetic and volumetric properties of a three-state protein folding system, comprising a metastable triple mutant of the Fyn SH3 domain, have been investigated using pressure-dependent 15N-relaxation dispersion NMR from 1 to 2500 bar. Changes in partial molar volumes (ΔV) and isothermal compressibilities (ΔκT) between all the states along the folding pathway have been determined to reasonable accuracy. The partial volume and isothermal compressibility of the folded state are ~100 ml·mol−1 and ~40 µl·mol−1·bar−1, respectively, higher than those of the unfolded ensemble. Of particular interest are the findings related to the energetic and volumetric properties of the on-pathway folding intermediate. While the latter is energetically close to the unfolded state, its volumetric properties are similar to those of the folded protein. The compressibility of the intermediate is larger than that of the folded state reflecting the less rigid nature of the former relative to the latter.

Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology

“…Similarly, Bezsonova et al implemented CPMG-type relaxation dispersion as a function of pressure to decompose the multistep folding pathway of an SH3 domain. Another relaxation dispersion study of a variant of the same SH3 domain exploited the full 2.5 kbar pressure range to collect an extensive dataset [27]. The thermodynamic values obtained are consistent with previously published values.…”

“…There are conflicting experimental measurements of the magnitude of Δβ° [13,17,20–23]. Moreover, experimental limitations most often preclude accurate measurement of the higher order terms [24–27] of the Taylor expansion and are usually assumed to be negligible.…”

Transition-State Compressibility and Activation Volume of Transient Protein Conformational Fluctuations

“…High-pressure NMR spectroscopy offers significant advantages, including a high-resolution view of protein structure and dynamics. 33 , 41 − 47 NMR studies have revealed the effect of pressure on the native-state protein structure 14 , 48 , 49 and its hydrogen bond network 44 , 46 as well as on protein-folding kinetics 42 , 50 − 54 and the pressure–temperature phase diagram of protein folding. 38 …”

General Expressions for Carr–Purcell–Meiboom–Gill Relaxation Dispersion for N-Site Chemical Exchange

“…Expressions for N -site ( N > 2) fast-limit exchange have been obtained from an eigenanalysis and applied in a linear three-site case . However, most analyses of linear three-site exchange in CPMG experiments have relied on numerical integration of the Bloch–McConnell equations. − …”