Spatially controlled assembly of enzyme conjugates can be applied to signal amplification systems for biosensors, or synthesis of novel catalysts for enzyme process technology. Artificial bienzymic systems comprised of two enzymes, NAD(P)H‐FMN oxidoreductase and luciferase (represented as blue and orange spheres, respectively, in the schematic representation) were assembled by means of DNA hybridization and streptavidin–biotin interaction.

High pressure NMR spectroscopy has developed into an important tool for studying conformational equilibria of proteins in solution. We have studied the amide proton and nitrogen chemical shifts of the 20 canonical amino acids X in the random-coil model peptide Ac-Gly-Gly-X-Ala-NH2, in a pressure range from 0.1 to 200 MPa, at a proton resonance frequency of 800 MHz. The obtained data allowed the determination of first and second order pressure coefficients with high accuracy at 283 K and pH 6.7. The mean first and second order pressure coefficients and for nitrogen are 2.91 ppm/GPa and −2.32 ppm/GPa2, respectively. The corresponding values and for the amide protons are 0.52 ppm/GPa and −0.41 ppm/GPa2. Residual dependent 1J1H15N-coupling constants are shown.

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The introduction of multidimensional NMR spectroscopy was a breakthrough in biological NMR methodology because it allowed the unequivocal correlation of different spin states of the system. The introduction of large pressure perturbations in the corresponding radio frequency (RF) pulse sequences adds an extra structural dimension into these experiments. We have developed a microprocessor-controlled pressure jump unit that is able to introduce fast, strong pressure changes at any point in the pulse sequences. Repetitive pressure changes of 80 MPa in the sample tube are thus feasible in less than 30 ms. Two general forms of these experiments are proposed here, the pressure perturbation transient state spectroscopy (PPTSS) and the pressure perturbation state correlation spectroscopy (PPSCS). PPTSS can be used to measure the rate constants and the activation energies and activation volumes for the transition between different conformational states including the folded and unfolded state of proteins, for polymerization–depolymerization processes, and for ligand binding at atomic resolution. PPSCS spectroscopy correlates the NMR parameters of different pressure-induced states of the system, thus allowing the measurement of properties of a given pressure induced state such as a folding intermediate in a different state, for example, the folded state. Selected examples for PPTSS and PPSCS spectroscopy are presented in this Article.

For a better understanding of nuclear magnetic resonance (NMR) detected pressure responses of folded as well as unstructured proteins the availability of data from well-defined model systems are indispensable. In this work we report the pressure dependence of chemical shifts of the backbone atoms (1)H(α), (13)C(α) and (13)C' in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of these nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The polynomial pressure coefficients B 1 and B 2 are dependent on the type of amino acid studied. The coefficients of a given nucleus 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 are also weakly correlated.

High-pressure (HP) NMR spectroscopy is an important method for detecting rare functional states of proteins by analyzing the pressure response of chemical shifts. However, for the analysis of the shifts it is mandatory to understand the origin of the observed pressure dependence. Here we present experimental HP NMR data on the (15) N-enriched peptide bond model, N-methylacetamide (NMA), in water, combined with quantum-chemical computations of the magnetic parameters using a pressure-sensitive solvation model. Theoretical analysis of NMA and the experimentally used internal reference standard 4,4-dimethyl-4-silapentane-1-sulfonic (DSS) reveal that a substantial part of observed shifts can be attributed to purely solvent-induced electronic polarization of the backbone. DSS is only marginally responsive to pressure changes and is therefore a reliable sensor for variations in the local magnetic field caused by pressure-induced changes of the magnetic susceptibility of the solvent.

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 C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH (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 and B is dependent on the type of atom and amino acid studied. For H, N and C the first order pressure coefficient B 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 stereospecificallyC enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.

NMR chemical shift analysis is a powerful method to investigate local changes in the environment of the observed nuclear spin of a polypeptide that are induced by application of high hydrostatic pressure. Usually, in the fast exchange regime, the pressure dependence of chemical shifts is analyzed by a second order Taylor expansion providing the first- and second-order pressure coefficient B1 and B2. The coefficients then are interpreted in a qualitative manner. We show here that in a two-state model, the ratio of B2/B1 is related to thermodynamic parameters, namely the ratio of the difference of compressibility factors Δβ' and partial molar volumes ΔV. The analysis is applied to the random-coil model peptides Ac-Gly-Gly-Xxx-Ala-NH2, with Xxx being one of the 20 proteinogenic amino acids. The analysis gives an average Δβ'/ΔV ratio of 1.6 GPa(-1) provided the condition |ΔG(0)| ≪ 2RT holds for the difference of the Gibbs free energies (ΔG(0)) of the two states at the temperature (T0) and the pressure (p0). The amide proton and nitrogen B2/B1 of a given amino acid Xxx are strongly correlated, indicating that their pressure-dependent chemical shift changes are due to the same thermodynamic process. As a possible physical mechanism providing a two-state model, the hydrogen bonding of water with the corresponding amide protein was simulated for isoleucine in position Xxx. The obtained free energy could satisfy the relation |ΔG(0)| ≪ 2RT. The derived relation was applied to the β-amyloid peptide Aβ and the phosphocarrier protein HPr from S. carnosus. For the transition of state 1 to state 2' of Aβ, the derived relation of B2/B1 to Δβ'/ΔV can be confirmed experimentally. The HPr protein is characterized by substantially higher negative B2/B1 values than those found in the tetrapeptides with an average value of approximately -5.1 GPa(-1) (Δβ'/ΔV of 5.1 GPa(-1) provided |ΔG(0)| ≪ 2RT holds). Qualitatively, the B2/B1 ratio can be used to predict regions of the HPr protein involved in the interaction with enzyme I or HPr-kinase/phosphatase.