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Pressure and protein dynamismKazuyuki Akasakaa

a High Pressure Protein Research Center, Institute of AdvancedTechnology, Kinki University, 930 Nishimitani, Kinokawa City,Wakayama 649-6493, JapanPublished online: 06 Apr 2014.

To cite this article: Kazuyuki Akasaka (2014) Pressure and protein dynamism, High PressureResearch: An International Journal, 34:2, 222-235, DOI: 10.1080/08957959.2014.882917

To link to this article: http://dx.doi.org/10.1080/08957959.2014.882917

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High Pressure Research, 2014Vol. 34, No. 2, 222–235, http://dx.doi.org/10.1080/08957959.2014.882917

Pressure and protein dynamism†

Kazuyuki Akasaka∗

High Pressure Protein Research Center, Institute of Advanced Technology, Kinki University, 930Nishimitani, Kinokawa City, Wakayama 649-6493, Japan

(Received 24 September 2013; final version received 9 January 2014)

The dynamism of life depends critically on the dynamism of bio-macromolecules themselves, notably pro-teins. The bio-macromolecular dynamism originates from weak non-bonding interactions, which inevitablyfluctuate under physiological conditions. The fascination lies in the fact that, in proteins, the basically ran-dom “non-biological fluctuations” of atoms are often turned into specific “biological fluctuations” of largeramplitude, which would be directly coupled to protein function and consequently the dynamism of life suchas growth, motility, sensing, adaptation and disease. The success of the combination of pressure perturba-tion with advanced nuclear magnetic resonance (NMR) spectroscopy by using the online cell method hasprovided 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 representativestrategies of high pressure NMR spectroscopy for characterizing protein dynamism will be discussed withactual examples.

Keywords: high pressure NMR; linear and nonlinear pressure shifts; high-energy conformers; biologicalfluctuation; dynamism of life

1. Introduction

1.1. Life and protein dynamism

Life is extremely dynamic and rich in variety. In spring, greens and flowers spring out fromthe darkness of the soil and change our surroundings dramatically into a rich mass of greensand colorful flowers, for example in a Japanese garden (Figure 1). Behind such dynamism oflife must be the dynamism of protein molecules that have been activated upon the change ofthe climate. For example, dihydrofolate reductase (DHFR) (Figure 1, right corner) is an enzymeessential for deoxyribonucleic acid (DNA) synthesis, catalyzing the reduction of 5,6-dihydrofolateto 5,6,7,8-tetrahydrofolate. A movie depicting conformational changes of DHFR during oneturnover of substrate has been constructed using six isomorphous crystallographic structures(URL: http://chem-faculty.ucsd.edu/kraut/dhfr.html).[1]

As this example shows, the macroscopic dynamism of life must has its basis in the dynamismof protein molecules, which carries the function. Knowing details of protein dynamics has thusbecome a central issue of life science.[2]

∗Email: akasaka@waka.kindai.ac.jp†This paper was presented at the LIth European High Pressure Research Group (EHPRG 51) Meeting at London (UK),1–6 September 2013.

© 2014 Taylor & Francis

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Figure 1. A dramatic change of a Japanese garden between winter and spring. Behind this drama is the dynamism ofproteins; for example, DHFR, an essential enzyme for DNA synthesis for all plants and animals.

1.2. Origins and characteristics of protein dynamism

How much do we know about protein dynamism? What are the specific questions to ask aboutit? Are there any unique features of it, different from the dynamism of all other materials such asmetals, plastics, polymers and lipids?

Protein is chemically a rather simple molecule, consisting mainly of atoms such as carbon,nitrogen, oxygen and hydrogen, but unique in that it is a functional molecule by itself. In contrastto the simpler digital aspect of the covalent-bonded primary (or chemical) structure directed bythe genetic code, the determination of its three-dimensional structure (folding) is an analog (orthermodynamic) process highly dependent on its environmental condition.[3]

Classically, a simple view has been that the protein folds into one unique structure called thenative structure, N, which is considered identical with the structure in crystal. This classical viewon the structure-functional relationship prevailed until recently, although small conceptual modi-fications of this simple view have been proposed in history such as the induced-fit model for ligandbinding by Koshland [4] and the allosteric concept for multi-domain proteins by Monod et al.[5]The classical concept of the protein structure with its limited dynamism must be replaced with amore realistic concept that proteins assume a large number of nearly isoenergetic conformations(conformational sub-states), as expressed by Frauenfelder, one of the founders of the modernconcept of proteins.[6] Under physiological conditions, these sub-states are frequently visited,contributing to the dynamism of proteins.

The dynamism, allowing environment-dependent fluctuations, originates from weak chemicalbonding among all atoms of the protein molecule and the surrounding water molecules. Theseinclude ionic interactions, interactions between partial atomic charges, hydrogen-bonding inter-actions, dihedral angle torsion potentials, van der Waals and London dispersion forces.[7] The

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224 K. Akasaka

inter-atomic potentials made up of these weak bonding interactions of non-biological naturefluctuate randomly and rapidly (<nsec) under physiological conditions, which form the basis forprotein dynamism.

1.3. Role of pressure perturbation

However, early investigations in the past decades have noticed that protein molecules may alsohave other mode of motions, which are more organized and apparently slower in time scale toperform specific functions in living cells, e.g. enzymatic reactions,[4] communications,[5] motil-ity, etc. Such characteristic dynamics of protein molecules, hereafter called “biological motions”,must have been developed by the life itself in the course of evolution over billions of years ofits long history on earth. When we speak about protein dynamics, we must be aware that weare facing the result of billions of years of history, by far the longest among all materials weindustrially make today.

There is a gap between the random atomic fluctuations which occur in the time scale of psec tonsec, and the more directed, cooperative fluctuations causing “biological motions” of the proteinmolecule that normally occur in μs–ms or even in seconds. To grasp the modern view of proteindynamism, we need to go on to characterize “biological motions” and correlate them with “non-biological fluctuations” of weak bonding potentials. The “biological motions” can be so subtle asto be slow in time scale (> μs) and involving extremely low-population conformers (<<10%)that a simple energetic or chemical perturbation such as high temperature or denaturant can easilysmear them out. We found that pressure perturbation of a few kbar (1 kbar = 100 MPa = 0.1 GPa;1 bar = 0.1 MPa = 0.9869 atm) can amplify such “biological motions” considerably through thevolumetric differences of the conformers involved without significantly changing their energylevels.[2]

2. Methods

2.1. Combination of multi-dimensional nuclear magnetic resonance spectroscopy withpressure

In nuclear magnetic resonance (NMR) spectroscopy, we need a relatively long time (ms–s) forrecording a free induction signal in the time domain, losing the trace of all rapid fluctuations(<<ms) in the resultant spectrum. On the other hand, slow fluctuations (>>ms) producing low-population conformers (<<10%) are usually not detectable either, as their signals are too weakto be recognized as conformers with separate signals.

To disclose the delicate and subtle dynamic balance between different sub-states of a proteinoccurring in the time scale of μs–s, we need to shift the balance systematically to visualizethe structure and the dynamics between the sub-states. Among other perturbations, pressure isthe best-suited from this point of view, and, in particular, when it is combined with an atomicresolution spectroscopy in solution, NMR, namely variable-pressure (or high pressure) NMRspectroscopy.[8]With this technique, one can explore the protein dynamism with little perturbationon their structures (except for slight compression), but affecting dramatically and systematicallythe equilibrium among the sub-states by thermodynamic perturbation. As a result, one can explorean exceptionally wider conformational space of a protein than hitherto explored, much beyondthe basic folded state, the main target explorable at 1 bar.[9]

The fluctuations of protein conformation, both rapid (<<ms) and slow (>>ms), which havebeen lost in conventional NMR spectroscopy, can be “recovered” in the high pressure NMR

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spectrum as pressure-induced changes in spectral parameters, e.g. chemical shift, signal intensity,J-coupling, line shape and spin relaxation. Combination of pressure at least up to a few knarwith 1H one- and two-dimensional and 15N/1H (or 13C/1H) hetero-nuclear two-dimensionalNMR measurements is frequently employed, the enrichment of proteins with 15N (or 13C) stableisotopes being required for the latter case. In the simple case, if assignments of 1H and/or 15Nsignals to specific protons and/or nitrogen have been made at ambient conditions prior to theapplication of high pressure NMR spectroscopy, it is usually possible to follow the changes ofindividual 1H and/or 15N signals (shifts and intensity) for specific proton and/or nitrogen signals,thereby giving the information as to the pressure-stabilized excited-state conformers.

2.2. Technical development in high pressure NMR

A history of development of high pressure NMR techniques is described elsewhere [8] and willbe only briefly touched with a more recent development. The undertaking of high pressure NMRmeasurement of proteins by placing the detection coil within the high pressure vessel (highpressure probe method) [10] demonstrated the high utility of high pressure NMR spectroscopy,but lacked the resolution required in modern structural biology. The success of the use of apressure-resistive sample cell, first made of synthetic quartz [11] on highly developed com-mercial NMR probes and spectrometers, promised the greater advancement of high pressureNMR application to proteins in later years. A recent development of ceramic (Zirconia) cells[12,13] is widening the utility of high pressure NMR spectroscopy to a larger community inprotein science because of its handiness and sensitivity,[14,15] although the method is still underdevelopment.

The advantage of the cell method combined with a commercial high-field NMR spectrometerover the pressure-vessel method is that the former can utilize essentially all the pulse sequencesincluding advanced techniques for two-dimensional and three-dimensional NMR measurements,essential for the site-specific structural information of proteins under pressure. However, the lattermethod retains the advantage of working in a higher pressure range (∼GPa) than the current limitof pressure of the cell method (up to 3 ∼ 4 kbar), and will continue to be useful for specificapplications.

In this report, high pressure NMR measurements were carried out on non-labeled, 15N-labeledand/or 13C-labeled proteins at variable pressure typically between 1 bar and 3.6 kbar. The onlinecell method was employed, with a pressure-resisting cell made of synthetic quartz with an innerdiameter of ∼1 mm and an outer diameter of ∼3 mm [11] at a proton frequency of 750 MHz orat 800 MHz.

3. Basic relations

3.1. “Elastic” response and “plastic” response of protein structures to pressure

In general, there are two ways of a protein in solution to attain a lower volume in response topressure; (a) by a general compression within the sub-ensemble of conformer, e.g. within N or I,and (b) by a transition (shift of conformational equilibrium) from a high-volume ensemble to alow-volume ensemble, e.g. from N to I.[16] Here, symbolically, we call the former process “Elasticresponse” and the latter process “Plastic response” (See Figure 2), although the distinction betweenthe two processes is not simple in proteins. [16] Here, the term “volume” refers to the partial molarvolume, i.e. the effective volume of the protein in solution (e.g. aqueous environment). In general,

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(a)

(b)

Figure 2. Illustration of the two ways of a protein in solution to attain a lower volume in response to pressure. (a)“Elastic” response by a compression within the sub-ensemble of a conformer, and (b) “Plastic” response by a transitionfrom a high-volume ensemble to a low-volume ensemble. Here, the term “volume” refers to the partial molar volume, i.e.the effective volume of the protein in water [Adapted from Figure 1 in [2] by permission of ACS Publications].

Scheme A is dominant at low pressure and Scheme B becomes dominant at elevated pressures,but, in principle, they occur simultaneously at different relative proportions.

3.2. Elastic response

Within the folded manifold of the protein molecule, the mean-square fluctuation of the volume< (δV)2 > of a protein at ambient pressure is intimately related to the compression of the volume(βV ) under pressure through the relation.[17]

< (δV)2 >= βVkT , (1)

where δV is the volume fluctuation, β is the isothermal compressibility coefficient, k is theBoltzmann’s constant and T is the absolute temperature.

Although, strictly speaking, this equation is valid only for macroscopic properties, it indicatesa likely relationship between local compressibility (i.e. inter-atomic volume changes) and localstructural fluctuation within the regime of the linear response theory. Our strategy is that wemeasure local compressibility of the protein molecule site-specifically with high-resolution highpressure NMR and interpret the result in terms of site-specific information on the conformationalfluctuation of the protein.

3.3. Plastic response

In general, a protein molecule in solution exists in a dynamic equilibrium mixture of sub-ensemblesof conformers differing, in general, in partial molar volume.[16] In a simple case of a two-stateequilibrium between the basic folded sub-ensemble N and an intermediately folded sub-ensembleI (cf. Figure 2), the protein differs both in thermodynamic stability (�G0 = G0(I) − G0(N)) andin partial molar volume (�V0 = V0(I) − V0(N)).

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In this situation, the equilibrium constant Kbetween N and I may change with pressure accordingto the following relationship:

K = [I][N] = exp

[−�G

RT

], (2)

where

�G = G(I) − G(N) = �G0 + �V0[p − p0] − [12

]�βV0[p − p0]2. (3)

Here �G and �G0 are the Gibbs-free energy changes from N to I at pressure p and p0(= 1 bar),respectively; �V0 is the partial molar volume change (negative for most proteins under physio-logical conditions); �β is the change in the compressibility coefficient; R is the gas constant andT is the absolute temperature.[16] Under physiological conditions, �G0 is normally positive (i.e.N is more stable than I) and the population of I is too low to be detected.

By applying pressure, the protein may assume a lower partial molar volume through a shift ofconformational equilibrium in favor of a lower volume conformer (sub-ensemble of conformers)(∼plastic response, Figure 2, b). For a typical case of �V0 = −20 ∼ −100 mL/mol, the �V0

(p − p0) value is on the order of −2 to −10 kJ/mol at 1 kbar and −4 to −20 kJ/mol at 2 kbar, whichmay be sufficient to bring the marginally positive �G0 to the negative side, thereby I becomingthe dominant species under pressure in place of N. This will enable the NMR measurement ofconformer I stably trapped under pressure and allow the analysis of its structure with establishedNMR techniques. When a protein consists of a series of conformers differing in volume, smallervolume conformers (namely a more disordered conformer) would populate at higher pressure,and, in principle, the NMR analysis of their respective conformers may be performed.

For some proteins, only fluctuations within the basic folded sub-ensemble N (elastic response,Figure 2(a)) may be monitored, while for many other proteins, one may explore the region ofplastic response, namely a significant part of their allowed conformational space beyond the foldedsub-ensemble N, including an intermediate conformer I (Figure 2(b)) and even those close to theunfolded conformer U.[18,19]

4. Applications

4.1. Elastic response within the folded sub-ensemble N, manifested by linear chemical shiftchanges of hen lysozyme

Microscopically, the elastic response of protein structure is best studied by analyzing pressure-induced chemical shift changes.[20]

4.1.1. Pressure-induced structural change in folded hen lysozyme

Pressure-induced chemical shift changes on diamagnetic protein systems were first measured onhen lysozyme using one-dimensional 1H NMR spectroscopy (Figure 3).[20] Pressure-inducedchanges of chemical shifts were measured for 26 protons of the protein with no loss of signalintensities, meaning that elastic response of the folded structure occurred, but no denaturationtook place up to 2 kbar of the hydrostatic pressure. The shifts were reversible and essentiallylinear with respect to pressure up to 2000 bar.

In a further study, we utilized two-dimensional 1H/1H Total correlated spectroscopy (TOCSY)and Nuclear Overhauser effect spectroscopy (NOESY) spectroscopy to extend the shift mea-surement to 240 protons, which showed essentially linear pressure dependence.[21] Structural

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(a) (b)

Figure 3. The pressure-dependent linear and reversible chemical shift changes in 1H one-dimensional NMR spectrum ofhen lysozyme in the pressure range of 30–2000 bar. Note: Measurements were made at pH 2, 20◦C at 800 MHz. Here, theapparent high-field shifts of some of the methyl proton signals with increasing pressure, immediately tell that a substantialcompaction takes place in the core part of the protein [Adapted from Figure 1 in [22] by permission of Elsevier].

restraints were generated as the change in chemical shift between 30 bar and 2 kbar, and, byincorporating the constraints into the X-PLOR force field, Williamson and his colleagues havedeveloped a strategy to calculate reliable and accurate structural changes from a high-qualitystarting structure.

Distances were calculated between all pairs of Cα atoms in the high pressure structure, andsubtracted from the corresponding distances in the low pressure structure. Although the changesinvolved are no more than 0.2Å, the resultant difference distance matrix gives a good indicationof regions that have moved relative to the rest of the protein (Figure 4).[21] Here, we note that

(1) The β-domain is compressed to a much larger extent than the α-domain, but the compressionoccurs quite heterogeneously over the entire tertiary structure of the protein.

(2) The largest changes in volume occur close to the buried water molecules and close to theligand-binding site, which are mainly located in the “hinge” region between the two domains.

(3) At 2 kbar, hydrogen bond lengths within the structure are reduced also quite heterogeneously;giving an average compaction by 0.011Å, but with a considerable standard deviation of0.107Å.

4.1.2. Cavities and hydration are the major sources of conformational fluctuation in henlysozyme

The resultant change in conformation at 2 kbar, calculated based on the experimental 1H chemicalshift changes (Figure 4), indicates that pressure causes a preferential compaction of the henlysozyme molecule centered around the water-containing cavities in the α−β inter-domain partand in part of the β domain. The result clearly indicates distinct structural variability or mobilityin residues surrounding the water-containing cavities.

The molecular dynamic simulation carried out on hen lysozyme at 1 bar (Figure 5, left) showsthat water molecules (represented by red dots) penetrate preferentially into the cavities.[22] Thewater-penetration sites coincide well with the sites for high compressibility (Figure 4). Combined,

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Figure 4. Elasticity of the hen lysozyme structure based on pressure-induced chemical shift changes. Note: Right:difference distance plot for Cα atoms of hen lysozyme in the basic folded state between 3 bar and 2 kbar. Orange andred contours denote parts of the structure that have moved closer together at 2 kbar, while cyan and blue contours denoteparts that have moved apart at 2 kbar. Secondary structure regions are indicated at the right side of the panel. Left:structural changes of hen lysozyme at 2 kbar, color coded to show regions of the structure that have moved most relativeto the rest of the structure. Data are average over all distances (d) between Cα atoms for each residue. Regions arecoded as: d < −0.12Å blue, −0.12 < d < −0.06 cyan, 0.16 < d < 0.22 orange and d > 0.22 red. The five buried watermolecules included in the calculation are shown in a red/pink space-filling representation [Adapted from Figure 1 in [21]by permission of Elsevier].

we may conclude that cavities and their hydration are the source of preferential mobility in henlysozyme. The fact that this occurs in the α−β inter-domain is consistent with the inter-domainmotions found in hen lysozyme,[23] where the catalytic reaction takes place, further indicatingthat such fluctuations may have been evolutionarily designed for the catalytic activity of thisprotein. On the other hand, the cavity hydration in the β domain could be important in directingthe folding pathway of this protein.[24]

4.1.3. Cavities are common in lysozymes

To further support the notion that the hydration of the cavity in the α−β inter-domain part isevolutionarily designed for the catalytic activity of this protein, the crystal structures of lysozymefrom eight different biological species are compared in Figure 6.[24] Surprisingly, we find that theircrystal structures are closely similar to each other, with their water-containing cavities conservedin the α−β inter-domain part in all animal species. The result gives no doubt about the criticalrole of the cavities in this family of proteins, for their function as well as folding.[21–24]

4.2. Plastic response beyond the folded sub-ensemble

4.2.1. Analysis of nonlinear chemical shifts

The nonlinear (or plastic) response of chemical shift to pressure has been found in almost allproteins other than the inhibitor proteins when pressure exceeds a few kbar (Figure 7)

In the data analysis, we use a Taylor expansion to the second order.[25] Namely, estimations oflinearity and nonlinearity of chemical shift changes with pressure are derived from least-square

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(a)

(b)

Figure 5. Water penetration into the interior of hen lysozyme shown by MD simulation. (a) Probability distribution (from0 to 10) of the total number of internally hydrated water molecules in hen lysozyme, the average being 4.81 (standarddeviation 1.61). (b) Penetration of water molecules into the protein interior obtained by molecular dynamic simulation at1 bar. Water molecules are shown by small red dots. Note: That water molecules penetrate preferentially into two specificsites where the cavities are located, namely in the hinge region between α and β domains and in the loop region of the β

domain [Adapted from Figure 2 in [22] by permission of Elsevier].

fits of experimental data for individual NMR signals to the following equation:

δi = ai + bip + cip2, (4)

where p is the pressure (GPa = 1000 MPa), δi the chemical shift (ppm) for the i-th residue, ai

(ppm) the chemical shift at zero pressure (≈0.1 MPa) and bi (ppm/GPa) and ci (ppm/GPa2)are the linear (first-order) and nonlinear (second-order) coefficients, respectively.[25] Here, thepressure dependence of the shift in the low pressure range is primarily determined by the bip term,while that in the high pressure range is determined more by the cip2 term.

4.2.2. Nonlinear response of chemical shifts provides a sensitive indicator of low-lying excitedstates

Nonlinear chemical shift response to pressure has been observed widely in globular proteins andthat in specific regions of three-dimensional structures (Figure 7, panels a–d).[26] Nonlinearityof the chemical shift would mean that the microscopic compressibility, given the chemical shiftchanges, varies with pressure. The variation of compressibility is expected when the protein

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Figure 6. Crystal structures (ribbon models) of lysozyme from eight different biological species. Note: PDB codes aregiven in parentheses. The water molecules trapped in cavities are shown by blue spheres [Adapted from Figure 7 of [26]by permission of Elsevier].

(a) (b) (c) (d)

(h)

(n)

Figure 7. Plots of amide 15N chemical shifts against pressure for selected residues in (a) BPTI, (b) ubiquitin, (c, h, n)NEDD8 and (d) HPr. (Residue numbers are shown in each panel). Panels h and n show 1H and 15N chemical shifts forVal 70 of ubiquitin [Adapted from Figure 10 and 19 in [26] by permission of Elsevier].

gradually assures a different conformational state(s), namely going into an excited state withincreasing pressure.

The degree of nonlinearity of pressure-induced chemical shift varies greatly from protein toprotein (Figure 8). We found earlier [25] that the magnitude of nonlinear shifts, averaged overall residues, varies greatly from protein to protein, while the magnitude of linear shifts, averagedover the molecule, does not vary much among the proteins. In general, for functional proteins,the nonlinearity or plasticity appears to represent the unique property of the protein more thanthe linearity or elasticity.[25] It is likely, therefore, that the plasticity, representing the propertyof low-lying excited states, is intimately related to the protein function, rather than the elasticity,representing mainly the property of the basic folded state.

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(a)

(f) (g) (h)

(j) (k) (l)

(n) (o) (p)

(m)

(i)

(b) (c) (d) (e)

Figure 8. Nonlinear pressure-induced 15N chemical shifts (�δ) expressed in the three-dimensional structures of 16 dif-ferent proteins. (a) Protein G, (b) BPTI, (c) ubiquitin, (d) NEDD8, (e) parkin-UBL, (f) HPr (WT), (g) HPr (H15A),(h) RasRBD, (i) RasRBD complex, (j) p13, (k) α-lactalbumin (MG), (l) lysozume, (m) prion, (n) apomyoglobin,(o) β-lactogloblin and (p) OspA. The extent of nonlinearity is shown by different colors (�δ > 20 ppm/GPa2 (red),15 < �δ < 20 ppm/GPa2 (orange) and 10 < �δ < 15 ppm/GPa2 (yellow), cf. Equation (4)) [Adapted from Figure 15in [26] by permission of Elsevier].

This expectation is fully borne out in ubiquitin, for which high pressure NMR study has beencarried out up to 3.6 kbar that showed a distinct sigmoidal shift with pressure, a clear evidence fora two-state like transition between the basic folded state and a low-lying excited state (Figure 7,panels h and n), as we see in more detail below.

4.2.3. Low pressure and high pressure conformations of ubiquitin

For ubiquitin, the first detailed structural analysis of an excited-state conformer was carried out onubiquitin at 3000 bar, disclosing structures of two major conformers (N and I) for ubiquitin (thelow pressure structure A and the high pressure structure B, Figure 11, top).[26] The low pressure

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(a) (b)

Figure 9. Top: (a) molecular surface of ubiquitin (pH 4.6, 20◦C) determined by conventional NMR at 30 bar'ilowpressure structure'jand (b) the same determined by high pressure NMR at 3 kbar (high pressure structure) [Adapted fromFigure 3,[28] by permission of Elsevier]. Bottom: Free energy levels of N and N’ conformers at low pressure and at highpressure (and transient conformer N*) based on MD simulation results [27] [Adapted from Figure 4 in [10] by permissionof ACS Publications].

structure is the well-known stable conformer (called “native” structure N), while the high pressurestructure is a new structure, the major difference from N is that N’ has an open structure in itscentral cleft. At any pressure, they are in dynamic equilibrium, N � N’, transforming to eachother in ∼10 μs time range.

An exceptionally long (μs) molecular dynamics simulation was carried out at 1 bar and 6000 barby Imai and Sugita.[27] They found that, while at low pressure the conformer N undergoes randomfluctuations in the psec–nsec range at all times within the N state itself, it undergoes occasionalfluctuations of larger amplitude and transforms into another ensemble of conformers, and back-and-forth. It turns out that this conformer is very close to what is found by high pressure NMR (N’in Figure 9, top). At high pressure, conformer N’ is stable undergoing rapid fluctuations in ∼nsec,but occasionally undergoes a large-amplitude fluctuation back to conformer N. In addition, theyfound additional states N* in between N and N’. Based on MD calculations, free energy diagramsare shown in Figure 9, bottom, for low pressure and for high pressure.

Intriguing is the finding that the larger amplitude transition occurs simultaneously with thepenetration of water molecules into the central cleft of the protein,[27] resulting in the loss ofvolume as experimentally observed (�V = −24 mL/mol [28]). In the conformer N’, most of

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the residues involved in the enzyme-binding region are exposed.[28] Thus it appears certain thatconformer I is a suitable design for the binding reaction with the E1 enzyme to take place, arequired step for its physiological function.

5. Conclusion

Dynamism of life, e.g. evolution, adaptation, growth, sensing, motion and disease, may appeardistant from atomic events in protein molecules, which originate simply from non-covalentinteractions among the atoms of the protein and those between the protein and the solvent (e.g.water). A crucial point here is the recognition that, in proteins, the psec–nsec random thermalfluctuations are occasionally turned into large-scale motions in the μs–ms time range (“biologicalfluctuations”), which are often directly coupled with function. Here, the role of high pressureNMR is to disclose and characterize such “biological fluctuations”, possibly with atomic-detailedensembles of excited-state conformers.

So far, limited pressure range and low sensitivity of measurement for many protein systems(like those reported in Figure 8) have not allowed detailed structural, thermodynamic and kineticanalyses of their excited-state conformers like that for ubiquitin. However, in the near future, whenthese and many other proteins become the target of detailed analysis by high pressure NMR, alongwith the development of high pressure crystallography [29], the concept of protein structure andits dynamism will largely be changed. Here we expect that a new era of bioscience will realize,in which the life dynamism and the molecular dynamism of proteins are no longer distant.

Acknowledgements

This work was supported by the Academic Frontier Program of the Ministry of Education, Culture, Sports, Science andTechnology, granted to Kinki University for 2007–2012.

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