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Small molecule binding within internal cavities provides a way to control protein function and structure, as exhibited in numerous natural and artificial settings. Unfortunately, most ways to identify suitable cavities require high-resolution structures a priori and may miss potential cryptic sites. Here we address this limitation via high-pressure solution NMR spectroscopy, taking advantage of the distinctive nonlinear pressure-induced chemical shift changes observed in proteins containing internal cavities and voids. We developed a method to rapidly characterize such nonlinearity among backbone 1H and 15N amide signals without needing to have sequence-specific chemical shift assignments, taking advantage of routinely available 15N-labeled samples, instrumentation, and 2D 1H/15N HSQC experiments. From such data, we find a strong correlation in the site-to-site variability in such nonlinearity with the total void volume within proteins, providing insights useful for prioritizing domains for ligand binding and indicating mode-of-action among such protein/ligand systems. We suggest that this approach provides a rapid and useful way to rapidly assess otherwise hidden dynamic architectures of protein that reflect fundamental properties associated with ligand binding and control.Significance StatementMany proteins can be regulated by internally binding small molecule ligands, but it is often not clear a priori which proteins are controllable in such a way. Here we describe a rapid method to address this challenge, using solution NMR spectroscopy to monitor the response of proteins to the application of high pressure. While the locations of NMR signals from most proteins respond to high pressure with linear chemical shift changes, proteins containing internal cavities that can bind small molecule ligands respond with easily identified non-linear changes. We demonstrate this approach on several proteins and protein/ligand complexes, suggesting that it has general utility.

β2-Microglobulin (β2m) is associated with dialysis-related amyloidosis. In vitro experiments have shown that β2m forms amyloid fibrils at acidic pHs in the presence of moderate concentrations of salt. Previous studies suggested that acid-denatured β2m has a hydrophobic residual structure, and the exposure of the hydrophobic residues enhances the association with seeds or other β2m monomers. However, the nature of the residual structure relevant to its amyloidogenicity remains to be investigated. To understand the structural properties of acid-denatured β2m and the role of salt, we investigated pressure- and salt concentration-dependent conformational changes by nuclear magnetic resonance spectroscopy and other methods. Here, pressure was utilized to characterize the conformers existing in a conformational equilibrium at ambient pressure. The obtained pressure- and salt concentration-dependent chemical shift data were simultaneously subjected to principal component analysis to characterize individual conformational change events. Unexpectedly, the addition of salt induced an expansion of the β2m molecule, which likely resulted from the exclusion of the N-terminal region from the hydrophobic cluster region. The dissected chemical shift patterns for the salt-induced conformational change and other experimental data indicated that this conformational change caused a rigidification in the intrinsic hydrophobic cluster, leading to the observed amyloidogenicity.

We have investigated conformational fluctuations in a new green fluorescent protein(GFP)-like protein rb-Akane found in a red-brown-colored octocoral, Scleronephthya gracillima (Kuekenthal)), with high pressure fluorescence spectroscopy at 0.1–700 MPa. Besides the green fluorescence at 510 nm, two red fluorescence peaks are observed at 590 and 629 nm, the relative intensity of which varies reversibly with pressure. The phenomenon is interpreted as representing the cis–trans isomerization of the chromophore accompanied by the conformational transition between two sub-states of the red fluorescence form of rb-Akane. The two sub-states are separated only marginally in free energy (ΔG0 = 1.9 ± 0.4 kJ mol−1), but significantly in partial molar volume (ΔV0 = −19.8 ± 1.4 ml mol−1) at 0.1 MPa (pH 7.5, 25°C). Above 500 MPa, the fluorescence at λmax 629 nm undergoes another reversible change with pressure, showing the onset of unfolding.

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Location: Plaza Verde, Numazu, Shizuoka, JapanDate: September 18th to 22nd, 2018After its inception in the year 1998, the international association of High Pressure Bioscience and Biotechnology (HP...

High-pressure NMR spectroscopy is a method that allows NMR measurements of any target samples under variable pressure. When applied to proteins, the method extends the conformational space that NMR spectroscopy can handle, from the space limited to the basic folded paradigm into the extensive conformational space that spans the entire high-energy paradigm of protein structure from the folded to the unfolded. The success of the experiment is ensured by the strong coupling between the volume and the conformation of a protein such that the partial molar volume of a protein decreases in parallel with the loss of its conformational order (the “volume theorem of protein”) as advocated by Akasaka. The validity of this principle arises from the fact that a globular protein generally has a significant vacant space (sometimes called “void” or “cavities”) inside its folded architecture into which water molecules may penetrate to reduce its partial molar volume and its conformational order simultaneously. Thanks to this principle, pressure provides a simple, clean, systematic and often efficient means of investigating the high-energy conformers of a protein, which is essential for understanding its folding, function, interaction, fibrillation, adaptation and evolution.

The dynamism of life depends critically on the dynamism of bio-macromolecules themselves, notably proteins. The bio-macromolecular dynamism originates from weak non-bonding interactions, which inevitably fluctuate under physiological conditions. The fascination lies in the fact that, in proteins, the basically random “non-biological fluctuations” of atoms are often turned into specific “biological fluctuations” of larger amplitude, which would be directly coupled to protein function and consequently the dynamism of life such as growth, motility, sensing, adaptation and disease. The success of the combination of pressure perturbation with advanced nuclear magnetic resonance (NMR) spectroscopy by using the online cell method has provided a powerful means for investigating details of such “biological fluctuations”, which encompass, in general, a much wider conformational space of a protein than hitherto explored. Some representative strategies of high pressure NMR spectroscopy for characterizing protein dyna...

The utility of tyrosine/tyrosinate fluorescence for pressure-unfolding studies of Trp-lacking proteins has been explored for the first time, with chicken ovomucoid (OVM) as target. A newly developed fluorescence spectrometer working in the range 0.1-700 MPa is employed for this purpose. At 25 °C at pH 12, all six Tyr residues give tyrosine emission at 306 nm, implying that all five Tyr residues are well buried at pH 12 in the folded OVM, except one giving "half-tyrosinate" emission at 325 nm. Upon increasing pressure, however, a distinct intermediate state, in which domains 1 and 2 are selectively unfolded, appears and increases up to 700 MPa. Extrapolated to 0.1 MPa, this intermediate lies 8.8±2.6 kJ mol(-1) above the native state, characterized with a partial molar volume smaller by -28.9±7.4 ml mol(-1). At 5 °C at 700 MPa, even domain 3 gives a sign of cold denaturation.

The unique role of pressure in protein folding studies is emphasized. Variable-pressure NMR experiments carried out under equilibrium conditions give unique opportunities to explore the energy landscape for protein folding. Intermediate conformers that may appear transiently in the kinetic folding experiments may be stably trapped under pressure, allowing examination of their conformations in site-specific detail with modern NMR spectroscopy. The intimate relationship between the kinetic folding experiment and the equilibrium pressure experiment is described with examples from ubiquitin and hen lysozyme.

Biomacromolecules are thermodynamic entities that exist in general as an equilibrium mixture of the basic folded state and various higher-energy substates including all functionally relevant ones. Under physiological conditions, however, the higher-energy substates are usually undetectable on spectroscopy, as their equilibrium populations are extremely low. Hydrostatic pressure gives a general solution to this problem. As proteins generally have smaller partial molar volumes in higher-energy states than in the basic folded state, pressure can shift the equilibrium toward the former substantially, and allows their direct detection and analysis with X-ray crystallography or NMR spectroscopy at elevated pressures. These techniques are now mature, and their status and selected applications are presented with future prospects.

We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml(-1) fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50-450 MPa the fluorescence intensity decreased rapidly with time (k(obs) = 0.00193 min(-1) at 0.1 MPa, 0.0348 min(-1) at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ~450 MPa, we determined the activation volume ΔV(0‡) = -32.9 ± 1.7 ml mol(monomer)(-1) and the activation compressibility Δκ(‡) = -0.0075 ± 0.0006 ml mol(monomer)(-1) bar(-1) for the dissociation reaction. The negative ΔV(0‡) and Δκ(‡) values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril.

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia call (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50 MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80 C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53 ml/mol at 25 degrees C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85 ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.

We applied 57Fe Mossbauer spectroscopy to determine occupation sites of Ni atoms in the hexagonal M-type strontium ferrite. From spectral analyses of Sr1 − x La x Fe11.6 − x Ni x O19 with x ≤ 0.35 at room temperature, we propose that Ni atoms occupy preferentially octahedral Fe (12k, 4f 2 and 2a) sites.

We have used low temperatures (down to -20°C) and high pressures (up to 2000 bar) to populate low-lying excited state conformers of hen lysozyme, and have analyzed their structures site-specifically using (15)N/(1)H two-dimensional HSQC NMR spectroscopy. The resonances of a number of residues were found to be selectively broadened, as the temperature was lowered at a pressure of 2000 bar. The resulting disappearance of cross-peaks includes those of residues in the β-domain of the protein and the cleft between the β- and α-domains, both located close to water-containing cavities. The results indicate that low-lying excited state conformers of hen lysozyme are characterized by slowly fluctuating local conformations around these cavities, attributed to the opportunities for water molecules to penetrate into the cavities. Furthermore, we have found that these water-containing cavities are conserved in similar positions in lysozymes from a range of different biological species, indicating that they are a common evolutionary feature of this family of enzymes.

NMR experiments at variable pressure reveal a wide range of conformation of a globular protein spanning from within the folded ensemble to the fully unfolded ensemble, herewith collectively called “high-energy conformers”. The observation of “high-energy conformers” in a wide variety of globular proteins has led to the “volume theorem”: the partial molar volume of a protein decreases with the decrease in its conformational order. Since “high-energy conformers” are intrinsically more reactive than the basic folded conformer, they could play decisive roles in all phenomena of proteins, namely function, environmental adaptation and misfolding. Based on the information on high-energy conformers and the rules on their partial volume in its monomeric state and amyloidosis, one may have a general view on what is happening on proteins under pressure. Moreover, one may even choose a high-energy conformer of a protein with pressure as variable for a particular purpose. Bridging “high-energy conformers” to macroscop...

The synthetic peptide fragment (LC5: LRCRNEKKRHRAVRLIFTI) inhibits human immunodeficiency virus type 1 (HIV-1) infection of MT-4 cells. In this study, the solution structure of LC5 in SDS micelles was elucidated by using the standard (1)H two-dimensional NMR spectroscopic method along with circular dichroism and fluorescence quenching. The peptide adopts a helical structure in the C-terminal region (residues 13-16), whereas the N-terminal part remains unstructured. The importance of Phe17 in maintaining the structure of LC5 was demonstrated by replacing Phe17 with Ala, which resulted in the dramatic conformational change of LC5. The solution structure of LC5 elucidated in the present work provides a basis for further study of the mechanism of the inhibition of HIV-1 infection.

There are some theoretical arguments related to interpreting the adiabatic compressibility (beta(s)) of a protein determined from the sound velocity and the difference between beta(s) and isothermal compressibility (beta(T)). To address these problems experimentally, we constructed a high-pressure oscillating densitometer and used it to measure the apparent specific volume of bovine serum albumin as a function of pressure (0.1-78 MPa) and temperature (5-35 degrees C). The beta(T) determined from plots of the apparent specific volume vs. pressure was slightly larger than beta(s) at all temperatures examined, with the difference between the two compressibilities increasing as the temperature was decreased. Only at room temperature did the observed beta(T) agree with those estimated from beta(s) using the heat capacity and the thermal expansibility of the protein, suggesting that there are significant as-yet-unknown mechanisms that affect protein compressibility.

In this work we measured 1H NMR chemical shifts for the ribonuclease barnase at pressures from 3 MPa to 200 MPa, both free and bound to d(CGAC). Shift changes with pressure were used as restraints to determine the change in structure with pressure. Free barnase is compressed by approximately 0.7%. The largest changes are on the ligand-binding face close to Lys-27, which is the recognition site for the cleaved phosphate bond. This part of the protein also contains the buried water molecules. In the presence of d(CGAC), the compressibility is reduced by approximately 70% and the region of structural change is altered: the ligand-binding face is now almost incompressible, whereas changes occur at the opposite face. Because compressibility is proportional to mean square volume fluctuation, we conclude that in free barnase, volume fluctuation is largest close to the active site, but when the inhibitor is bound, the fluctuations become much smaller and are located mainly on the opposite face. The timescale of the fluctuations is nanoseconds to microseconds, consistent with the degree of ordering required for the fluctuations, which are intermediate between rapid uncorrelated side-chain dynamics and slow conformational transitions. The high-pressure technique is therefore useful for characterizing motions on this relatively inaccessible timescale.

Bovine plasma albumin Fr. V (BPA) has been known to contain small amounts of proteolytic enzyme. Wilson & Foster (1971) found a very limited and specific cleavage of BPA catalyzed by the enzyme with BPA in the F-form near pH 3.8, resulting in the formation of partially hydrolyzed BPA (BPA*). BPA* had a tendency to form a transparent gel at pD 4.0 (pD range of the F-form) above 8%, though proteolytic enzyme-free bovine mercaptalbumin (BMA) was in a transparent solution at pD 4.0 even at 12%. Water structures of the F-form of BMA in the solution state and of BPA* in the gel state were studied by measuring 1H-n.m.r. spectra, spin-lattice relaxation time (T1) and cross relaxation time (TIS) between irradiated and observed protons. Protein concentration-dependent changes of T1 of water protons indicated that the amount of hydrated water of BPA* in the gel state is far greater than that of the F-form of BMA in the solution state. TIS values from protein protons to water protons also indicated a large amount of hydration of BPA*, strong interaction between water and BPA* and rapid exchange between bound and bulk water in the gel state.

The acid-induced isomerization (the N-F transition) and expansion of sodium dodecyl sulfate-bovine plasma albumin complex (ADm; m, molar ratio of added sodium dodecyl sulfate to bovine plasma albumin; O less than or equal to m less than or equal to 12) were studied by measuring CD-resolved secondary structure, fluorescence polarization and life-time of tryptophyl fluorophors, acid-titration with the electrostatic correction for the surface potential, 1H-n.m.r. spectra and cross relaxation time between irradiated and observed protons. The immobilization of tryptophyl fluorophors observed in the F-form of AD0 was suppressed in the F-form of AD10. The acidtitration analysis of AD12 showed non salt-bonding between carboxylate groups and cationic side chains in the F-form, as in the case of AD0, indicating charged side chains being presumably mobile. 1H-n.m.r. spectra and cross relaxation times between irradiated and observed protons in the F-form of AD10 indicated the increase in the local motion. On the other hand, AD10 and AD12 did not show any significant change in the CD-resolved secondary structure in the N-F transition region. The F-form of AD10 or AD12 may therefore be the moltenglobule state which has secondary structure similar to the N-form of the complexes with fluctuating tertiary structure (side chains).

The equilibrium unfolding of hen lysozyme at pH 2 was studied as a function of pressure (0.1~700 MPa) and temperature (−10°C~50°C) using Trp fluorescence as monitor supplemented by variable pressure 1H NMR spectroscopy (0.1~400 MPa). 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 ΔG (=GU−GN) were evaluated from changes in the wavelength of maximum fluorescence intensity (λmax) as a function of pressure and temperature. The dependence of ΔG on temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (ΔCp=CpU−CpN=1.8−1.9 kJ mol–1 deg–1) at all pressures studied (250~400 MPa), while the temperature TS for maximal ΔG increased from about 10°C at 250 MPa to about 40°C at 550 MPa. The dependence of ΔG on pressure gave negative volume changes (ΔV=VU−VN) upon unfolding at all temperatures studied (−86~−17 ml mol–1 for −10°C~50°C), which increase significantly with increasing temperature, giving a positive expansivity change (Δα~1.07 ml mol–1 deg–1). A phase-diagram between N and U (for ΔG=0) is drawn of hen lysozyme at pH 2 on the pressure-temperature plane. Finally, a three-dimensional free energy landscape (ΔG) is presented on the p-T plane.

A 20-residue peptide E5 containing five glutamates, an analog of the fusion peptide of influenza virus hemagglutinin (HA) exhibiting fusion activity at acidic pH lower than 6.0-6.5 was studied by circular dichroism (CD), Fourier transform infrared, and 1H-NMR spectroscopy in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine (DPC) micelles, and phospholipid vesicles. E5 became structurally ordered at pH < or = 6 and the helical content in the peptide increased in the row: water < water/TFE < DPC approximately = phospholipid vesicle while the amount of beta-structure was approximately reverse. 1H-NMR data and line-broadening effect of 5-, 16-doxylstearates on proton resonances of DPC bound peptide showed E5 forms amphiphilic alpha-helix in residues 2-18, which is flexible in 11-18 part. The analysis of the proton chemical shifts of DPC bound and CD intensity at 220 nm of phospholipid bound E5 showed that the pH dependence of helical content is characterized by the same pKa approximately 5.6. Only Glu11 and Glu15 in DPC bound peptide showed such elevated pKas, presumably due to transient hydrogen bond(s) Glu11 (Glu15) deltaCOO- (H+)...HN Glu15 that dispose(s) the side chain of Glu11 (Glu15) residue(s) close to the micelle/water interface. These glutamates are present in the HA-fusion peptide and the experimental half-maximal pH of fusion for HA and E5 peptides is approximately 5.6. Therefore, a specific anchorage of these peptides onto membrane necessary for fusion is likely driven by the protonation of the carboxylate group of Glu11 (Glu15) residue(s) participating in transient hydrogen bond(s).

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small-angle X-ray scattering (SAXS) and 1H-NMR spectroscopy. Our results indicate that the urea-induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3 degrees C) occur at least in two steps. In the urea concentration range of 0-2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2-4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.

The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (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 GPa(-1) at 278 K and 2.20 ppm GPa(-1), respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa(-1). 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 beta-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 beta-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.

Study of the effects of pressure on macromolecular\ud structure improves our understanding of the forces\ud governing structure, provides details on the relevance\ud of cavities and packing in structure, increases\ud our understanding of hydration and provides a basis\ud to understand the biology of high-pressure organisms.\ud A study of DNA, in particular, helps us to\ud understand how pressure can affect gene activity.\ud Here we present the first high-resolution experimental\ud study of B-DNA structure at high pressure, using\ud NMR data acquired at pressures up to 200 MPa\ud (2 kbar). The structure of DNA compresses very\ud little, but is distorted so as to widen the minor\ud groove, and to compress hydrogen bonds, with AT\ud pairs compressing more than GC pairs. The minor\ud groove changes are suggested to lead to a compression\ud of the hydration water in the minor groove.

Remarkable acceleration of enzymatic proteolysis by pressure at kbar range is reported with ubiquitin as substrate and α-chymotrypsin as enzyme. The acceleration is interpreted in terms of the shift of conformational equilibrium in ubiquitin from the non-degradable folded conformer to the enzyme-degradable unfolded conformer by pressure because of the lower volume of the latter, while the enzymatic activity of α-chymotrypsin is still largely retained. This mechanism is considered generally applicable to most globular proteins and the method of pressure-accelerated proteolysis will have an enormous potential utility in systems wherever efficient removal of proteins is needed.

The solution structure of the GB1 domain of protein G at a pressure of 2 kbar is presented. The structure was calculated as a change from an energy-minimised low-pressure structure using (1)H chemical shifts. Two separate changes can be characterised: a compression/distortion, which is linear with pressure; and a stabilisation of an alternative folded state. On application of pressure, linear chemical shift changes reveal that the backbone structure changes by about 0.2 A root mean square, and is compressed by about 1% overall. The alpha-helix compresses, particularly at the C-terminal end, and moves toward the beta-sheet, while the beta-sheet is twisted, with the corners closest to the alpha-helix curling up towards it. The largest changes in structure are along the second beta-strand, which becomes more twisted. This strand is where the protein binds to IgG. Curved chemical shift changes with pressure indicate that high pressure also populates an alternative structure with a distortion towards the C-terminal end of the helix, which is likely to be caused by insertion of a water molecule.

We report here results of the first direct measurement of partial volume and compressibility changes of a protein as it forms an amyloid protofibril. We use a high precision density meter and an ultrasonic velocity meter on a solution of intrinsically denatured, disulfide-deficient variant of hen lysozyme, and follow the time-dependent changes in volume and compressibility, as the protein spontaneously forms a protofibril. We have found a large increase in partial specific volume with time from 0.684 to 0.724 mL x g-1 (Deltanu = 0.040 mL x g-1 corresponding to 570 mL x (mol monomer)-1) and in partial specific adiabatic compressibility coefficient from -7.48 x 10(-12) to +1.35 x 10(-12) cm2 x dyn-1 (Deltabetas = 8.83 x 10(-12) x cm2 x dyn-1) as the monomer transforms into a protofibril. The results demonstrate that the protofibril is a highly voluminous and compressible entity, disclosing a cavity-rich, fluctuating nature for the amyloid protofibril. The volume and compressibility changes occur in two phases, the faster one preceding the major development of the beta-structure in the protofibril as monitored by CD.

Protein structure in solution fluctuates over a wide range of conformation, but the reality of the fluctuation has been little explored. In this article, we present an approach to this problem using variable pressure NMR spectroscopy. The method enables us to explore protein conformation over a wide range of conformational space than hitherto explored by conventional NMR method and brings about a new dynamic view on protein structure. The highlight of the method is the determination of the structural coordinates of semi-stable higher energy conformers that exist in low population in dynamic equilibrium with the stable "native" conformer under physiological conditions. Available evidence suggests that such high energy conformers play crucial roles in function in many proteins.

The combination of high-resolution NMR spectroscopy with pressure perturbation, known as variable-pressure NMR spectroscopy or simply high pressure NMR spectroscopy, is a relatively recent accomplishment, but is a technique expanding rapidly with high promise in future. The importance of the method is that it allows, for the first time in history, a systematic means of detecting and analyzing the structures and thermodynamic stability of high-energy sub-states in proteins. High-energy sub-states have been only vaguely known so far, as normally their populations are too low to be detected by conventional spectroscopic techniques including NMR spectroscopy. By now, however, high pressure NMR spectroscopy has established unequivocally that high-energy conformers are universally present in proteins in equilibrium with their stable folded counterparts. This chapter describes briefly the techniques of high pressure NMR spectroscopy and its unique and novel aspects as a method to explore protein structure in its high-energy paradigm with illustrative examples. It is now well established that high pressure NMR spectroscopy is a method to study intrinsic fluctuations of proteins, rather than those forced by pressure, by detecting structural changes amplified by pressure. Extension of the method to other bio-macromolecular systems is considered fairly straightforward.

The combination of fluorescence and pressure perturbation is a widely used technique to study the effect of pressure on a protein system to obtain thermodynamic, structural and kinetic information on proteins. However, we often encounter the situation where the available pressure range up to 400 MPa of most commercial high-pressure fluorescence spectrometers is insufficient for studying highly pressure-stable proteins like inhibitors and allergenic proteins. To overcome the difficulty, we have recently developed a new high-pressure fluorescence system that allows fluorescence measurements up to 700 MPa. Here we describe the basic design of the apparatus and its application to study structural and thermodynamic properties of a couple of highly stable allergenic proteins, hen lysozyme and ovomucoid, using Tryptophan and Tyrosine/Tyrosinate fluorescence, respectively. Finally, we discuss the utility and the limitation of Trp and Tyr fluorescence. We discuss pitfalls of fluorescence technique and importance of simultaneous use of other high-pressure spectroscopy, particularly high-pressure NMR spectroscopy.

The conformational fluctuation in the minimum DNA-binding domain of c-Myb, repeats 2 and 3 (R2R3), was studied under closely physiological conditions. A global unfolding transition, involving both the main chain and the side chains, was found to take place at the approximate temperature range 30-70 °C, with a transition temperature of approximately 50 °C. In addition, the observation of simultaneous shift change and broadening of NMR signals in both (1)H one-dimensional and (15)N/(1)H two-dimensional NMR spectra indicated the occurrence of locally fluctuating state at physiological temperature. In the wild-type protein containing a cavity in R2, the local fluctuation of R2 is more prominent than that of R3, whereas it is suppressed in the cavity-filled mutant, V103L. This indicates that the cavity in R2 contributes significantly to the conformational instability and the transition into the locally fluctuating state. For the wild-type R2R3 protein, the more dynamic conformer is estimated to be present to some extent at 37 °C and is likely beneficial for its biological function: DNA-binding. This result is in agreement with the concept of an excited-state conformer that exists in equilibrium with the dominant ground-state conformer and acts as the functional conformer of the protein. From the findings of the present study, it appears that the tandem repeats of two small domains with no disulfide bonds and with a destabilizing cavity function as the evolutionary strategy of the wide-type c-Myb DNA-binding domain to produce an appropriate fraction of the locally fluctuating state at 37 °C, which is more amenable to DNA-binding. Database: Chemical shifts and peak lists have been deposited in the Biological Magnetic Resonance Bank under entries 11584 and 11585.

A theory to describe structural fluctuations of protein induced by thermodynamic perturbations, pressure, temperature, and denaturant, is proposed. The theory is formulated based on the three methods in the statistical mechanics: the generalized Langevin theory, the linear response theory, and the three dimensional interaction site model (3D-RISM) theory. The theory clarifies how the change in thermodynamic conditions, or a macroscopic perturbation, induces the conformational fluctuation, which is a microscopic property. The theoretical results are applied, on the conceptual basis, to explain the experimental finding by Akasaka et al., concerning the NMR experiment which states that the conformational change induced by pressure corresponds to structural fluctuations occurring in the ambient condition. A method to evaluate the structural fluctuation induced by pressure is also suggested by means of the 3D-RISM and the site-site Kirkwood-Buff theories.

The investigation of common structural motifs provides additional information on why proteins conserve similar topologies yet may have non-conserved amino acid sequences. Proteins containing the ubiquitin superfold have similar topologies, although the sequence conservation is rather poor. Here, we present novel similarities and differences between the proteins ubiquitin and NEDD8. They have 57% identical sequence, almost identical backbone topology and similar functional strategy, although their physiological functions are mutually different. Using variable pressure NMR spectroscopy, we found that the two proteins have similar conformational fluctuation in the evolutionary conserved enzyme-binding region and contain a structurally similar locally disordered conformer (I) in equilibrium with the basic folded conformer (N). A notable difference between the two proteins is that the equilibrium population of I is far greater for NEDD8 (DeltaG(0)(NI)5 kJ/mol) than for ubiquitin (DeltaG(0)(NI)=15.2(+/-1.0) kJ/mol), and that the tendency for overall unfolding (U) is also far higher for NEDD8 (DeltaG(0)(NU)=11.0(+/-1.5) kJ/mol) than for ubiquitin (DeltaG(0)(NU)=31.3(+/-4.7) kJ/mol). These results suggest that the marked differences in thermodynamic stabilities of the locally disordered conformer (I) and the overall unfolding species (U) are a key to determine the functional differences of the two structurally similar proteins in physiology.

With the high-resolution variable-pressure NMR spectroscopy, one can study conformational fluctuations of proteins in a much wider conformational space than hitherto explored by NMR and other spectroscopic techniques. This is because a protein in solution generally exists as a dynamic mixture of conformers mutually differing in partial molar volume, and pressure can select the population of a conformer according to its relative volume. In this review, we describe how variable-pressure NMR can be used to probe conformational fluctuations of proteins in a wide conformational space from the folded to the fully unfolded structures, with actual examples. Furthermore, the newly emerging technique "NMR snapshots" expresses amply fluctuating protein structures as changes in atomic coordinates. Finally, the concept of conformational fluctuation is extended to include intermolecular association leading to amyloidosis.

A protein in solution is a thermodynamic entity existing in equilibria among multiple conformations from the folded to the unfolded. Various experimental results indicate that transitions to higher energy conformers are crucial for function in many proteins. However, our knowledge on higher energy conformers is extremely limited at present, compared to the knowledge about the folded counterparts. A newly emerging technique, the variable pressure NMR method, enables us to explore a much wider conformational space of proteins than hitherto explored by X-ray crystallography and conventional NMR technique, giving a new dynamic view of proteins that amply fluctuate over the entire conformational space.

Extensive structural studies using high-pressure NMR spectroscopy have recently been carried out on proteins, which potentially contribute to our understanding of the mechanisms of protein folding. Pressure shifts the conformational equilibrium from higher to lower volume conformers. If the pressure is varied, starting from the folded native structure, in many cases we observe intermediate conformers before the onset of total unfolding. This enables the investigation of details of the structure and thermodynamic characteristics of various intermediate conformers of proteins under equilibrium conditions. We can also examine pressure effects on the structure and stability of some typical denatured states such as helical denatured, molten globule, and unfolded states. The high-pressure NMR method can also be used to investigate association/dissociation equilibria of oligomeric or aggregated proteins. Beside direct observation of kinetic intermediates upon pressure jump, NMR structural investigations of equilibrium conformers under pressure provide information about the structures of kinetic intermediates during folding/unfolding reactions.

Although a diversity of proteins is known to form amyloid fibers, their common mechanisms are not clear. Here, we show that an intrinsically unfolded protein (U), represented by a disulfide-deficient variant of hen lysozyme with no tertiary structure, forms an amyloid-like fibril after prolonged incubation. Using variable pressure NMR along with sedimentation velocity, circular dichroism, and fluorescence measurements, we show that, before the fibril formation, the protein forms a pressure-dissociable, soluble assemblage ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{U}}_{{\mathrm{n}}}^{^{\prime}}\end{equation*}\end{document} ) with a sedimentation coefficient of 17 S and a rich intermolecular β-sheet structure. The reversible assemblage is characterized with a Gibbs energy for association of –23.3 ± 0.8 kJ·mol –1 and a volume increase of 52.7 ± 11.3 ml·mol –1 per monomer unit, and involves preferential interaction of hydrophobic residues in the initial association step. These results indicate that amyloid fibril formation can proceed from an intrinsically denatured protein and suggest a scheme \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{N}}{\Leftrightarrow}{\mathrm{U}}{\Leftrightarrow}{\mathrm{U}}_{{\mathrm{n}}}^{^{\prime}}{\rightarrow}{\mathrm{fibril}}\end{equation*}\end{document} as a common mechanism of fibril formation in amyloidogenic proteins, where two-way arrows represent reversible processes, one-way arrow represents an irreversible process, and N, U, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{U}}_{{\mathrm{n}}}^{^{\prime}}\end{equation*}\end{document} represent, respectively, the native conformer, the unfolded monomeric conformer, and the soluble assemblage of unfolded conformers.