Use of high pressure for the study of
conformational states and dynamics in biological systems


Observation of intermediate states of the human prion protein by high pressure NMR spectroscopy
Norman Kachel, Werner Kremer, Ralph Zahn and Hans Robert Kalbitzer

Background: Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrPSc, of the cellular prion protein, PrPC. The conversion and thus the propensity of PrPC to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physicochemical properties of proteins as well as the dynamics and structure of folding intermediates.
Results: Conformational intermediates of the human prion protein huPrPC were characterized by a combination of hydrostatic pressure (up to 200 MPa) with two-dimensional NMR spectroscopy. All pressure effects showed to be reversible and there is virtually no difference in the overall pressure response between the folded core of the N-terminal truncated huPrPC(121–230) and the full-length huPrPC(23–230). The only significant differences in the pressure response of full-length and truncated PrP suggest that E168, H187, T192, E207, E211 and Y226 are involved in a transient interaction with the unfolded N-terminus. High-pressure NMR spectroscopy indicates that the folded core of the human prion protein occurs in two structural states N1and N2 in solution associated with rather small differences in free enthalpies (3.0 kJ/mol). At atmospheric pressure approximately 29% of the protein are already in the pressure favored conformation N2. There is a second process representing two possible folding intermediates I1 and I2 with corresponding average free enthalpies of 10.8 and 18.6 kJ/mol. They could represent preaggregation states of the protein that coexist at ambient pressure with a very small population of approximately 1.2% and less than 0.1%. Further the pressure response of the N-terminus indicates that four different regions are in a fast equilibrium with non-random structural states whose populations are shifted by pressure.
Conclusion: We identified pressure stabilized folding intermediates of the human prion protein. The regions reflecting most strongly the transition to the intermediate states are the ß1/a1-loop and the solvent exposed side of a3. The most pressure-sensitive region (representing mainly intermediate I1) is the loop between ß-strand 1 and a-helix 1 (residue 139–141), indicating that this region might be the first entry point for the infectious conformer to convert the cellular protein.
Pressure-induced local unfolding of the Ras binding domain of RalGDS
Kyoko Inoue, Hiroaki Yamada, Kazuyuki Akasaka, Christian Herrmann, Werner Kremer, Till Maurer, Rolf Döker and Hans Robert Kalbitzer
The reliable prediction of the precise three-dimensional structure of proteins from their amino acid sequence is a major, still unresolved problem in biochemistry. Pressure is a parameter that controls folding/unfolding transitions of proteins through the volume change ÄV of the protein-solvent system. By varying the pressure from 30 to 2,000 bar we detected using 15N/1H 2D NMR spectroscopy a unique equilibrium unfolding intermediate I in the Ras binding domain of the Ral guanine nucleotide dissociation stimulator (Ral GDS). It is characterized by a local melting of specific structural elements near hydrophobic cavities while the overall folded structure is maintained.
15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure
HR Kalbitzer, A Gorler, H Li, PV Dubovskii, W Hengstenberg, C Kowolik, H Yamada and K Akasaka

The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by twodimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa21 at 278 K and 2.20 ppm GPa21, respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa21. Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the b-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded b-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.
High-pressure NMR study of the complex of a GTPase Rap1A with its effector RalGDS
Kyoko Inouea, Till Maurer, Hiroaki Yamada, Christian Herrmann, Gudrun Horn, Hans Robert Kalbitzer, Kazuyuki Akasaka
Abstract Unusually large non-linear 1H and 15N nuclear magnetic resonance chemical shifts against pressure have been detected for individual amide groups of the Ras-binding domain of Ral guanine dissociation stimulator (GDS). The non-linear response is largest in the region of the protein remote from the Rap1A-binding site, which increases by about two-fold by the complex formation with its effector protein Rap1A. The unusual non-linearity is explained by the increasing population of another conformer (N'), lying energetically above the basic native conformer (N), at higher pressure. It is considered likely that the conformational change from N to NP in the Ras-binding domain of RalGDS works as a switch to transmit the effector signal further to molecules of different RalGDS-dependent signaling pathways.
Infrequent cavity-forming fluctuations in HPr from Staphylococcus carnosus revealed by pressure- and temperature-dependent tyrosine ring flips
Mineyuki Hattori, Hua Li, Hiroaki Yamada, Kazuyuki Akasaka, Wolfgang Hengstenberg, Wolfram Gronwald and Hans Robert Kalbitzer

Infrequent structural fluctuations of a globular protein is seldom detected and studied in detail. One tyrosine ring of HPr from Staphylococcus carnosus, an 88-residue phosphocarrier protein with no disulfide bonds, undergoes a very slow ring flip, the pressure and temperature dependence of which is studied in detail using the on-line cell high-pressure nuclear magnetic resonance technique in the pressure range from 3 MPa to 200 MPa and in the temperature range from 257 K to 313 K. The ring of Tyr6 is buried sandwiched between a b-sheet and a-helices (the water-accessible area is less than 0.26 nm²), its hydroxyl proton being involved in an internal hydrogen bond. The ring flip rates 10^1~­10^5 1/s were determined from the line shape analysis of HD1, HD2 and HE1, HE2 of Tyr6, giving an activation volume dV of 0.044 ± 0.008 nm³ (27 mL/mol), an activation enthalpy dH of 89 ± 10 kJ/mol, and an activation entropy dS of 16 ± 2 J/(K mol). The dV and dH values for HPr found previously for Tyr and Phe ring flips of BPTI and cytochrome c fall within the range of dV of 28 to 51 mL/mol and dH of 71 to 155 kJ/mol. The fairly common dV and dH values are considered to represent the extra space or cavity required for the ring flip and the extra energy required to create a cavity, respectively, in the core part of a globular protein. Nearly complete cold denaturation was found to take place at 200 MPa and 257 K independently from the ring reorientation process.
High-sensitivity sapphire cells for high pressure NMR spectroscopy on proteins
Martin Reinhard Arnold, Hans Robert Kalbitzer and Werner Kremer
High pressure NMR spectroscopy is a most exciting method for studying the structural anisotropy and conformational dynamics of proteins. The restricted volume of the high pressure glass cells causes a poor signal to noise ratio which up to now renders the application of most of the multidimensional NMR experiments impossible. The method presented here using high strength single crystal sapphire cells doubles the signal-to-noise ratio and allows to perform high pressure NMR measurements more easily. As a first application the difference of partial molar volumes caused by cis–trans-isomerisation of a prolyl peptide bond in the tetrapeptide Gly-Gly-Pro-Ala could be determined as 0:25ml/mol at 305 K.

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