Abstract
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 1H and 15N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (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 B1 and B2 that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the 1H and 15N 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 Hα and HN 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.
Highlights
The study of the pressure response of polypeptides and proteins by high pressure NMR spectroscopy can be used for characterization of the free energy landscape of proteins
By applying pressure to the tetrapeptides and fitting the resulting pressure dependence of the chemical shift to Eq 1, we obtained a complete dataset of 1H and 15N random coil chemical shift values for side chains of the amino acid 3 in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2
When comparing the spectra of isotope labeled with the corresponding unlabeled amino acids, a difficulty is the isotope shift that occurs in the SAIL amino acids since these are stereoselectively deuterated and 15N and 13C enriched
Summary
The study of the pressure response of polypeptides and proteins by high pressure NMR spectroscopy can be used for characterization of the free energy landscape of proteins (for reviews see e.g. Kitahara et al 2013; Akasaka and Matsuki 2015). The pressure response allows the detection of rare “excited” conformational states of proteins that are important for folding and function For the detection of rare states of proteins, mainly pressure dependent changes of chemical shifts are evaluated that are in many cases non-linear and can often be fitted with an appropriate thermodynamical model. These conformational contributions have to be separated from other chemical shift contributions as they are observed even in non-folded model compounds as a consequence of compression and rearrangement of the water shell.
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