Solution NMR investigation of the response of the lactose repressor core domain dimer to hydrostatic pressure

Abstract

Previous investigations of the sensitivity of the lac repressor to high-hydrostatic pressure have led to varying conclusions. Here high-pressure solution NMR spectroscopy is used to provide an atomic level view of the pressure induced structural transition of the lactose repressor regulatory domain (LacI* RD) bound to the ligand IPTG. As the pressure is raised from ambient to 3 kbar the native state of the protein is converted to a partially unfolded form. Estimates of rotational correlation times using transverse optimized relaxation indicates that a monomeric state is never reached and that the predominate form of the LacI* RD is dimeric throughout this pressure change. Spectral analysis suggests that the pressure-induced transition is localized and is associated with a volume change of approximately −115 ml/mol and an average pressure dependent change in compressibility of approximately 30 ml mol−1 kbar−1. In addition, a subset of resonances emerge at high-pressures indicating the presence of a non-native but folded alternate state.

High-Pressure NMR Reveals Volume and Compressibility Differences Between Two Stably Folded Protein Conformations

“…Pressure directly affects protein conformational equilibria by acting on differences in the partial volumes and compressibilities of different conformers, with increasing pressure favoring conformations with lower volumes 21 . As such, pressure tends to denature proteins as unfolded proteins are generally smaller than their folded forms; such transitions typically occur above 2000 bar under native conditions [20][21][22][23][24][25] . However, the application of sub-unfolding pressures can often cause proteins to shift populations among multiple folded states 18,[26][27][28] , providing an easily applied (and removed) way to manipulate protein conformational equilibria to facilitate their study.…”

Reverse Micelle Encapsulation of Proteins for NMR Spectroscopy

“…( D ) 125 μM 15 N-MBP (41 kDa) in 75 mM 10MAG/LDAO (65:35 molar percent ratio), 30 mM hexanol, 20% D-pentane, W 0 = 12 with 5500 psi pressure. IL-1β and LacI ae unstable under pressure in ethane due to the pressure sensitivity of LacI to unfolding and monomerization (Fuglestad, Stetz, Belnavis, & Wand, 2017) and pressure sensitivity of IL-1β from an internal hydrophobic cavity (Quillin, Wingfield, & Matthews, 2006). Spectral signal for both of these proteins is abrogated within 24 hours.…”

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

“…The change in isothermal compressibility of unfolding (Δβ°) represents the difference in the compressibility of the native and the unfolded states. 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.…”

“…Solution NMR spectroscopy at room temperature has complemented, often recapitulated, and greatly extended these findings [41]. As detailed below, chemical shift analysis can reveal population redistributions between the native state and nearby alternative states or locally unfolded states [13,23,42]. Protein dynamics studies as a function of pressure have illustrated the large and heterogeneous response of side chains that accompany the more subtle structural changes [43,44].…”

“…For example, alternative states in intermediate exchange would be undetectable, chemical shifts of different probes can report on different physical phenomena, and the magnitude of the coefficients in δ(p) do not need to correlate with the thermodynamic properties of the exchanging states, including Δβ Nevertheless, chemical shift changes can also report on the physical contributors to the compressibility of proteins such as the existence of multiple states in the folded ensemble that are sensitive to pressure, including locally unfolded states, and on the broadening or narrowing of the energy well of a “single” (the native) state. In particular, it has been found that proteins in which large cavities were engineered yield larger B 2 values, suggestive of a wider ensemble of accessible conformations [11,23,32,42,85].…”