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For interpreting the pressure induced shifts of resonance lines of folded as well as unfolded proteins the availability of data from well-defined model systems is indispensable. Here, we report the pressure dependence of 1 H and 15 N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH 2 (Xxx is one of the 20 canonical amino acids) measured at 800 MHz proton frequency. As observed earlier for other nuclei the chemical shifts of the side chain nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The pressure response is described by a second degree polynomial with the pressure coefficients B 1 and B 2 that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the 1 H and 15 N resonances of the guanidine group of arginine. In addition, stereoselectively isotope labeled SAIL amino acids were used to support the stereochemical assignments. The random-coil pressure coefficients are also dependent on the neighbor in the sequence as an analysis of the data shows. For H α and H N correction factors for different amino acids were derived. In addition, a simple correction of compression effects in thermodynamic analysis of structural transitions in proteins was derived on the basis of random-coil pressure coefficients.

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 3kbar 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 -115mlmol -1 and an average pressure dependent change in compressibility of approximately 30mlmol -1 kbar -1 . In addition, a subset of resonances emerge at high-pressures indicating the presence of a non-native but folded alternate state., (Copyright © 2017 Elsevier B.V. All rights reserved.)

For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of 13 C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH 2 (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The size of the polynomial pressure coefficients B 1 and B 2 is dependent on the type of atom and amino acid studied. For H N , N and C α the first order pressure coefficient B 1 is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically 13 C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.

The equilibrium unfolding of hen lysozyme at pH 2 was studied as a function of pressure (0.1~700MPa) and temperature (-10°C~50°C) using Trp fluorescence as monitor supplemented by variable pressure 1 H NMR spectroscopy (0.1~400MPa). The unfolding profiles monitored by the two methods allowed the two-state equilibrium analysis between the folded (N) and unfolded (U) conformers. The free energy differences Δ G (= G U - G N ) were evaluated from changes in the wavelength of maximum fluorescence intensity ( λ max ) as a function of pressure and temperature. The dependence of Δ G on temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (Δ C p = C p U - C p N = 1.8-1.9 kJ mol -1 deg -1 ) at all pressures studied (250~400 MPa), while the temperature T S for maximal Δ G increased from about 10°C at 250MPa to about 40°C at 550MPa. The dependence of Δ G on pressure gave negative volume changes (Δ V = V U - V N ) upon unfolding at all temperatures studied (-86~-17 mlmol -1 for -10°C~50°C), which increase significantly with increasing temperature, giving a positive expansivity change (Δ α ~1.07mlmol -1 deg -1 ). A phase-diagram between N and U (for Δ G =0) is drawn of hen lysozyme at pH 2 on the pressure-temperature plane. Finally, a three-dimensional free energy landscape (Δ G ) is presented on the p-T plane.