Preserving Proteins Under High Pressure and Low Temperature

Our group has a continuing interest in devising new freezing-protection agents. To this end, we have systematically investigated the structure of aqueous electrolyte salt solutions and room-temperature ionic liquids (RTILs), which consist of the cations and anions. We found that such water-mixed solutions remain liquid even at 1.0 GPa and 77 K (achieved by rapid cooling at 500 K/min) (Imai et al., 2010; Takekiyo et al., 2006a, 2006b; Yoshimura et al., 2009, 2011) and that water-soluble RTILs such as 1-butyl-3-methylimidazolium tetrafluoroborate remain liquid even at 2.0 GPa (Imai et al., 2011, Takekiyo et al., 2011). These results suggest that high pressure and low temperature by the use of RTILs-water mixtures might be useful for the freezing preservation of aqueous protein solutions.


Introduction
A promising method of preserving aqueous protein solutions is to subject them to high pressure and low temperature.However, two significant issues can arise under these conditions: (1) ice nucleation in aqueous solution at 273 K and 1.0 GPa, and (2) structural changes to the protein (Kunugi & Tanaka, 2002;Frank, 1995;Heremans & Smeller, 1998;Vandekooi, 1998).
The first issue can be avoided by addition of a freezing-protection agent such as sugar or salt.For example, Miyazaki et al. reported that myoglobin in a highly aqueous NaCl solution forms a glassy state during quenching and annealing processes (Miyazaki et al., 1993(Miyazaki et al., , 2000)).
Our group has a continuing interest in devising new freezing-protection agents.To this end, we have systematically investigated the structure of aqueous electrolyte salt solutions and room-temperature ionic liquids (RTILs), which consist of the cations and anions.We found that such water-mixed solutions remain liquid even at 1.0 GPa and 77 K (achieved by rapid cooling at 500 K/min) (Imai et al., 2010;Takekiyo et al., 2006aTakekiyo et al., , 2006b;;Yoshimura et al., 2009Yoshimura et al., , 2011) ) and that water-soluble RTILs such as 1-butyl-3-methylimidazolium tetrafluoroborate remain liquid even at 2.0 GPa (Imai et al., 2011, Takekiyo et al., 2011).These results suggest that high pressure and low temperature by the use of RTILs-water mixtures might be useful for the freezing preservation of aqueous protein solutions.
The second issue can best be addressed by developing a better understanding of the structural stability of aqueous protein solutions under conditions of high pressure and low temperature.
Pressure-temperature (P-T) phase diagrams provide valuable information about the structural stability of proteins.Such diagrams have been obtained by thermodynamic analysis of the pressure and temperature dependencies of structural stability by various experimental methods including NMR, UV-VIS, and vibrational spectroscopy (Brandts et al., 1970;Hawley, 1971;Lassale et al. 2000;Panick et al., 2005;Taniguchi & Suzuli, 1983;Yamaguchi et al., 1995;Zip & Kauzman, 1973).They show that, for many proteins, pressures of >0.5 GPa induce pressure unfolding and temperatures of <253 K induce cold unfolding.
With respect to pressure unfolding, many proteins-such as ribonuclease A(RNase A), lysozyme, myoglobin, and trypsin-pressure unfold at >0.5 GPa (Meersman et al, 2002(Meersman et al, , 2003;;Ruan et al., 1999;Smeller et al., 2006), whereas other proteins such as cytochrome c and trp repressor do not pressure unfold under high pressure.Dewa et al. and Dubins et al. showed that cytochrome c undergoes pressure-induced recovery (Dewa et al., 1998, Dubins et al., 2003).Moreover, Desai et al. showed that the population of the solvated -helical structure of trp repressor increases from approximately 20% to 40% with increasing pressure, whereas the population of the buried -helical structure, located in the interior of the protein, decreases from approximately 60% to 30% (Desai, et al., 1999).
With respect to cold unfolding, Nash and Jonas suggest that, for some proteins, the coldunfolded state might resemble an early folding intermediate (Nash & Jonas, 1997).Zhang et al. showed that the cold-unfolded state retains a native-like core structure (Zhang et al., 1995).Building on their results, Meersman et al. reported that cold unfolding results in the formation of a partially unfolded state in which some secondary contacts are still present (Meersman et al, 2002a).
These results suggest the following: Under high pressure, helical proteins such as myoglobin and cytochrome c exhibit different structural stabilities, and / -and -proteins such as RNase A, lysozyme, and trypsin tend to unfold in water.At low temperature, protein denaturation induces partial unfolding.Thus, two important questions arise: What dominant factor accounts for the different structural stabilities of -proteins under high pressure?And what is the nature of the structural changes induced in proteins at low temperature?Our goal, in investigating the feasibility of protein preservation under high pressure and low temperature, is to answer these questions.To this end, we report herein the systematic investigation of pressure-and temperature-induced changes in the secondary structures of oligopeptides and helical proteins by means of FTIR and circular dichroism (CD) spectroscopy combined with density functional theory (DFT) and geometric volume calculations.

Samples
For peptide samples, Ac-YGAA(KAAAA) 2 KA-NH 2 (AK peptide) (Shimizu Laboratory, Soka University) and poly-L -glutamic acid (PLGA) sodium salts (MW = 8000; Peptide Inc.) were used without further purification.For protein samples, bovine serum albumin (BSA) and horse-heart cytochrome c (Sigma-Aldrich Co.) were used without further purification.Figure 1 shows the three-dimensional structures of these peptide and proteins.Sample solutions were prepared as follows.For FTIR spectral measurements, solutions were prepared at concentrations of 20 mg/mL in two different buffer solutions (20 mM MES, pD 5.0; 50 mM tris-DCl buffer, pD 7.6).All exchangeable backbone amide protons in the peptides and proteins were deuterated by incubating in a D 2 O solution at 298 K. Completion of hydrogen-deuterium exchange was confirmed by the cessation of shifts in the amide II band.This band, in the frequency region around 1550 cm  1 , is known to shift to around 1450 cm  1 on deuteration of the backbone amide protons.For CD spectral measurements, solutions were prepared at concentrations of 0.1 mg/mL (~0.06 mM) in a buffer solution (pD 6.7).Sample concentrations were determined by UV absorption at 280 nm of Tyr and Trp (ε = 1197 M  1 cm  1 for Tyr and 5559 M  1 cm  1 for Trp).

High-pressure and low-temperature experiments
FTIR and CD spectroscopy are powerful tools for investigating changes in the secondary structures of peptides and proteins.For our purposes, the amide I' (the deuterium peptide groups) vibrational mode, which consists of C=O stretching, C-N stretching, and C-C-N deformation in the region 1620-1690 cm  1 (Figure 2), is highly sensitive to the secondary structures of peptides and proteins, and thus serves as an indicator of -helical and/orsheet structures (Bandekar, 1992;Krimm & Bandekar, 1986).Similarly, CD spectroscopy enables estimation of the helical content of proteins.CD-ellipticity at 208 and 222 nm, which originates from n-π* excitation of the peptide backbone, correlates strongly with helical content (Chen et al., 1974;Greenfield et al., 1969;Kelly et al., 2005).We previously reported the relevant details of FTIR and CD spectroscopy (Takekiyo et al., 2006c(Takekiyo et al., , 2009)).For high-pressure experiments, we used a diamond anvil cell (DAC) (Figure 3a).Sample solution was placed together with a small amount of -quartz for a pressure marker (Siminovitch, et al., 1987;Wong, et al., 1985) in a Hasteloy C-276 gasket (φ = 1.0 mm, t = 0.05 mm) mounted in the DAC (Figure 3b).Pressure was measured to a precision of ±0.05 GPa.The infrared beam was condensed by a zinc selenide lens system onto the sample in the DAC.For low-temperature experiments, we used a cryostat chamber (Figure 4a).The outer windows of the chamber were made of CaF 2 .A Teflon spacer (100 μm) was placed between two CaF 2 windows in the transmission cell (Figure 4b), the cell was set in the chamber, and the chamber was filled with liquid N 2 at 0.1 MPa.Temperature was controlled with a mass flow controller.The cooling speed was 30 K/min.
Geometric volume calculation has been conducted as the analysis methodology for obtaining the information on a relationship between the partial molar volume (PMV) and the structure of a biomolecule (Imai et al., 2005a(Imai et al., , 2005b;;Ling et al., 1998).The geometric volume calculations of peptides and proteins were carried out using the alpha-shapes program (Edelsbrunner et al., 1995;Ling et al., 1998).Geometric volume component of peptides and proteins enables the calculation of the solvent accessibility of the molecular surface, assuming that a water molecule is a hard-sphere probe (Edelsbrunner et al., 1995;Imai et al., 2005aImai et al., , 2005b;;Ling et al., 1998).In the present study, we calculated both van der Waals volume (V w ) and molecular volume (V M ).The latter, V M , consists of V w plus void volume (V v ), which is the volume of the structural void within the solvent-inaccessible core of the solute molecule.Hence, V v can be obtained by subtracting The diameter of water molecule was taken as 2.928 Å, again assuming that a water molecule is a hard-sphere (Imai et al., 2005a).Figure 5 shows schematic definitions of these various volume contributions.

Pressure-induced structural stability of proteins
Figure 6 shows FTIR and second-derivative spectra as a function of pressure in the amide I' region for AK peptide and PLGA.The spectra show three peaks at 1635, 1645, and 1672 cm  1 for AK peptide and three peaks at 1637, 1648, and 1670 cm  1 for PLGA.In a previous FTIR study (Manas et al., 2000;Reisdorf et al., 1996;Silva et al., 2002, Walsh et al., 2003), two peaks at 1635 and 1637 cm  1 for AK peptide and PLGA were assigned to the solvatedhelical structure, as shown in the same figure.The peak frequency for a solvated -helical structure is lower than the characteristic frequency for a buried -helical structure (~1650 cm  1 ) due to the hydrogen bond between the -helix and water molecule as shown in Fig. 7.The peak at ~1645 cm  1 for both peptides is thus assigned to a random-coil structure.The peaks at 1672 cm  1 for AK peptide are assigned to the asymmetric carboxylate stretching of trifluoroacetic acid ion remained after dialysis (Williams et al., 1996).The peak at 1670 cm  1 for PLGA is assigned to a turn structure.Significantly, for AK peptide and PLGA, the solvated -helical structures increase with pressure but the random-coil structures decrease from the second derivative spectra.Thus, the solvated -helical structures of AK peptide and PLGA are clearly stabilized at high pressure.Similar observations have previously been reported.For example, Desai et al.
reported that the solvated -helical structure of trp repressor is maintained at 0.8 GPa (Desai et al., 1999).A recent FTIR study showed that the solvated -helical structures of alaninebased peptide (Imamura et al., 2009), four-helix bundle protein ( -l-) 2 (Takekiyo et al., 2006c) and GCN4-p1 (Imamura et al., 2010) do not unfold even at >1.0 GPa; rather, with increasing pressure, the populations of the buried -helical structure decrease and those of the solvated -helical structure increase, implying that the solvated -helical structures of ( -l-) 2 and GCN4-p1 hydrate strongly under high pressure.In contrast, however, Meersman et al. reported that the -helical structure of myoglobin begins to break down at >0.5 GPa and denatures completely at 0.7 GPa (Meersman et al., 2002), and Wojciech et al.
reported that apo and holo types of -lactalbumin unfold completely at 0.5 GPa (Wojciech et al., 1999).Thus, from the viewpoint of the pressure-induced unfolding of helical proteins, the structural stabilities of even similar helical proteins can differ from one another under high pressure.

Pressure effect on amide I' frequency shifts of proteins: Interpretation from dft calculations
What, then, is the dominant factor affecting the structural stability of helical proteins under high pressure?We investigated this question by examining the amide I' frequency shift and geometric volume.The pressure-induced amide I' frequency shift of a protein is clearly related to environmental changes around the protein.
Recent FTIR studies of helical peptides and proteins have shown that, for ( -l-) 2 and GCN4-p1, with increasing pressure, the amide I' frequency of the solvated -helical structures (~1635 cm  1 ) shift to lower frequency (Takekiyo et al., 2006c;Imamura et al., 2010).Our current results are consistent with these findings.Figure 8 shows that, for AK peptide and PLGA, with increasing pressure, both amide I' frequencies of the solvatedhelical structures shift to lower frequency.
To clarify the nature of this pressure-induced amide I' frequency shift for the -helical structures of proteins, we focus on the hydrogen bond (H-bond): the intermolecular H-bond between the -helical structure and water molecules.We then explain the pressure-induced shift to lower frequency by speculating that, with increasing pressure, the intermolecular Hbond between the C=O group of the peptide bond and water molecules strengthens, the C=O force constant weakens, and the contribution of C=O bond unharmonicity increases.
As a model system of solvated -helical structure, we selected N-methylacetamide (NMA; CH 3 CONHCH 3 ) dimer + D 2 O complex.NMA is the simplest model compound for investigating the structural and physical properties of the peptide group (Ham et al. 2003;Kubelka & Keiderling, 2001).For our present calculations for the NMA dimer + D 2 O complex, we examine the two peaks at 1632 and 1656 cm  1 , which are amide I' peaks arising from the NMA-D 2 O H-bond and NMA H-bond free, respectively (Figure 9).The former Hbond pattern corresponds to the solvated -helical structure.

Origin of pressure stability of proteins: Interpretation from geometric volume calculations
We now consider the pressure stability of proteins in terms of geometric volume.PMV is a fundamental thermodynamic quantity that characterizes protein conformation (Chalikian, 2003) and is of principal importance in the analysis of pressure-induced denaturation of protein by Le Chatelier's law (Balny et al., 2002;Royer, 2002).Assuming that the protein unfolding process is a two-state transition (folded (F) ↔ unfolded (U)), the relationship between the pressure dependence of the equilibrium constant (K) and PMV is as follows: where K is the equilibrium constant, where ΔV is the difference in PMV between the protein's folded and unfolded states.From this relationship, it is evident that increasing pressure causes the protein structure to change so as to minimize PMV (ΔV < 0).Therefore, analysis of PMV differences associated with pressure unfolding can improve our understanding of the structural stability of proteins under high pressure.
Drawing upon the PMV analysis reported by Chalikian and Breslauer (Chalikian & Breslauer, 1996), we now systematically consider PMV differences between the folded and unfolded conformers of helical peptides and proteins.The PMV difference between the folded and unfolded states can be decomposed into three terms as follows: where ΔV w and ΔV V are the van der Waals and void-volume differences, respectively, and ΔV Hyd is the hydration volume difference caused by changes in intermolecular interactions between the solvent water and solute.
We suggest that protein void space may be the most significant factor affecting the pressure stability of proteins.Lopez et al. have speculated similarly (Lopes et al., 2004).They investigated the pressure dependence of the volume contribution of the hydrophobic internal cavity inside three-helix bundle mutant proteins and showed that a large cavity space induces a large change in volume.Therefore, we focus herein on the void volume of various helical peptides and proteins to elucidate the relationship between the pressure stability and volume properties of proteins.
We systematically calculated the geometric volumes of various peptides and proteins (probe size 2.98 Å).Table 1 shows the volume properties of various peptides and proteins.Figure 11(a) shows plots of V w , V M , and V v as a function of molecular weight (MW).V w , V M , and V v all increase linearly with MW.Imai et al. recently calculated the MW dependence of the molecular volume of various proteins and showed that V v correlates well with MW (Imai et al., 2005a); our results are consistent with these.Figure 11(b) shows plots of V v /MW as a function of MW.V v /MW increases up to MW = ~20000 and remains constant thereafter.
The pressure stability of a protein structure is thought to correlate with the adiabatic compressibility ( ) of the folded protein (Taulier & Chalikian, 2002;Gekko, 2004).That is, in a folded protein, increasing V v causes an increase in .The values for the well-known helical proteins cytochrome c (Kamiyama et al., 1999) and myoglobin (Gekko & Noguchi, 1974) are 3.60 and 8.98 Mbar  1 , respectively.The former is clearly smaller than the latter.
Similarly, the V v /MW values for cytochrome c and myoglobin are 0.153 and 0.223 cm 3 •mol −1 MW  1 , respectively.Thus, for these two proteins, the difference in relationship of V v /MW values correlates with the difference in values.Next, we consider the pressure stability of proteins obtained by our present and previous results.The helical structures of AK peptide (15 residues), helix bundle proteins (64 residues) (Takekiyo et al., 2006c), and GCN4-P1 (68 residues) (Imamura et al., 2010) do not unfold even at ~1.0 GPa.The V v /MW values for these helical peptides and proteins are all <0.2 cm 3 •mol −1 •MW −1 .In contrast, the helical structures of myoglobin (153 residues) (Meersman et al., 2002) and -lactalbumin unfold completely at 0.7 GPa (Wojciech et al., 1999).The V v /MW value for myoglobin is 0.223 cm 3 •mol −1 •MW −1 .From Figure 11 (b), we see that the structures of helical peptides and proteins for which V v /MW < 0.2 cm 3 ･mol -1 ･MW -1 exhibit small pressure stability, and those for which V v /MW > 0.2 cm 3 ･mol -1 ･MW -1 exhibit large pressure stability.Thus, our calculation result is in qualitatively good agreement with previous experimental results for the pressure stability of helical peptides and proteins, and both the examined proteins do indeed exhibit large pressure stability.On the basis of these calculations, we suggest that helical proteins having a small void space are stabilized at high pressure.
Our findings so far suggest that, for successful preservation under high pressure, two conditions must be met: (1) the -helical structure must be sufficiently hydrated with water molecules, and (2) the protein must have a small void space.

Comparisons of the structural stability of proteins at low temperature
We have just discussed the structural stability of proteins under high pressure.We now focus on the structural stability of proteins at low temperature (liquid N 2 , 77 K).
Figure 12 shows the FTIR (upper) and CD (lower) spectra of PLGA, BSA, and cytochrome c in aqueous solution at 298 K and 77 K, both at 0.1 MPa.The FTIR spectrum of PLGA shows an intriguing change: the peak at 1638 cm  1 increases in intensity with decreasing temperature from 298 to 77 K.This peak position is close to that assigned to the solvatedhelical structure, despite the fact that bulk water is crystallized at 77 K.If low temperature induces the solvated -helical structure of PLGA, the associated CD spectrum at low temperature should show double negative bands at 208 and 222 nm, characteristic of anhelical structure.Indeed, the CD spectra of PLGA show, at 298 K, a negative band at 195 nm, characteristic of a random-coil structure, the CD spectra show the characteristic of -helical structure.Thus, low temperature induces the transition of PLGA from coil to helix and then stabilizes the resulting solvated -helical structure.
What is the -helical structure of proteins at 77 K? In Figure 12, the peaks for BSA and cytochrome c at ~1635 cm  1 increase with decreasing temperature from 298 to 77 K.The CD spectra show negative bands at 208 nm and 222 nm even at 77 K, albeit at changed intensity.Thus, the solvated -helical structure of PLGA, BSA, and cytochrome c is clearly stabilized at 77 K despite the crystallization of bulk water.Moreover, these spectral changes are completely reversible.Thus, the low-temperature-induced stabilities of the solvatedhelical structure of helical proteins are very similar to the pressure-induced stabilities.
Previous FTIR studies by Manas et al. and Walsh et al. showed that the solvated -helical structure of 3 D (a de novo designed three-helix bundle protein), GCN4-p1, and parvalbumin in glycerol-D 2 O mixed solution does not unfold at low temperature (10 K) but rather is stabilized (Manas et al., 2000;Walsh et al., 2003).Generally, addition of glycerol to a protein solution is known to induce incremental structural stability in the protein (Zelent et al., 2004;Wright et al., 2003).Our present results show that the solvated -helical structures of PLGA, BSA, and cytochrome c are stabilized at 77 K without the addition of glycerol.On the other hand, the secondary structures of RNase A, lysozyme, and trypsin having a -sheet structure show an increase in the ratio of unfolded structure to -sheet structure at 77 K (data not shown).Based on these results, we found the difference of the structural stability of proteins at 77 K from the viewpoint of two secondary structure types (the -helix andsheet).A general rule is that the solvated -helical structure is hydrophilic and the -sheet structure is hydrophobic.Typically, the stability of -sheet cores in proteins requires the exclusion of water since the hydrogen bonding is much sparser between the -sheets (between individual strands) than between the turn or -helix.If the bulk water is frozen it is impossible for the water exclusion effects to act and no reduction in free energy is achieved by formation of -sheet structure.In contrast, as stated above, -helical structures are formed by H-bonds between successive loops in the helix.These H-bonds would come more stable (relative to thermal fluctuations) at low temperature.Hence, lowering the temperature may even induce -helix formation such as PLGA and helical proteins.
In this section, our findings so far suggest that, for successful preservation under low pressure, one condition must be met: the -helical structure must be sufficiently hydrated with water molecules as in the case of high pressure.

Feasibility of protein preservation under high pressure and low temperature
As mentioned in Section 1, a necessary condition for protein preservation under high pressure and low temperature is that ice formation must be avoided, and this can be accomplished by addition of a freezing-protection agent such as sugar or salt.For example, Yamamoto et al. recently reported compression recovery of the secondary structure of RNase A in a sucrose-D 2 O mixed solution (Yamamoto, et al. 2010).Manas et al. and Walsh et al. reported, by FTIR study, that the secondary structures of 3 D, GCN4-p1, and parvalbumin in a glycerol-D 2 O mixed solution do not unfold at low temperature (10 K), suggesting that the solvated -helical structure hydrated with water molecules is stabilized under this condition (Manas et al., 2000;Walsh et al., 2003).Moreover, Thus, under high pressure and low temperature, sugar solutions can induce retention of the secondary structures of proteins.
Given that the protein structure can be retained with addition of a freezing-protection agent, can it also be retained without addition of such an agent?Our present and previous results (Meersman et al, 2002(Meersman et al, , 2003;;Ruan et al., 1999;Smeller et al., 2006) show that proteins having a -sheet structure unfold at high pressure and show an increase in the ratio of unfolded structure to -sheet structure at 77 K. Therefore, protein preservation under high pressure and low temperature is difficult without the use of a freezing-protection agent.However, our present results show that the secondary structures of helical proteins having a small void space do not unfold at high pressure and low temperature.Thus, helical proteins may resist low-temperature-and high-pressure-induced protein unfolding, and their preservation under high-pressure and low-temperature conditions may thus be feasible.

Summary and conclusions
Protein preservation under high pressure and low temperature can be hampered by two issues: (1) conversion of bulk water to ice, and (2) structural instability of the protein by pressure unfolding (which occurs at pressures of >0.5 GPa) and cold unfolding (which occurs at temperatures of <243 K).The first issue can be solved by use of a freezingprotection agent.Our present results show that the second issue is not a concern for helical proteins having a small void space because these proteins are clearly stabilized at high pressure (~1.0 GPa) or low temperature (77 K).Thus, proteins having a high degree of solvated -helicity have a high feasibility of preservation under high pressure and low temperature, even without the use of a freezing-protection agent.
Many previous studies have reported the structural stability of proteins in aqueous sugar or salt solutions at low temperature, but only a few have done so at high pressure.However, both pressure and temperature play critical roles in regulating the structures and properties of proteins, and both are important tools for exploring new methods of protein preservation.Our present results demonstrate the feasibility of protein preservation under conditions of both high pressure and low temperature.

Fig. 2 .
Fig. 2. Relationship between the amide I and I' modes of protein.

Fig. 3 .
Fig. 3. (a) Schematic picture of diamond anvil cell (DAC) and (b) Schematic diagram of diamond anvils with a gasket and -quartz.

Fig. 6 .Fig. 7 .
Fig. 6.FTIR (upper) and second derivative (lower) spectra in the amide I' region of (a) AK peptide and (b) PLGA in aqueous solution as a function of pressure.

Fig. 8 .Fig. 9 .
Fig. 8.The peak wavenumber of the solvated -helical structure of AK peptide and PLGA in aqueous solution as a function of pressure.The closed and open circles represent PLGA and AK peptide, respectively.

Fig. 10 .
Fig. 10.Changes in the amide I' frequency of NMA in the NMA-dimer + D 2 O complex as a function of the bond distance (d O ･･･ O ) between the C=O group of the peptide bond of NMA dimmer and the O-D group of water calculated at B3LYP/6-311+(d,p) basis set.

Fig
Fig. 11.(a) Fig. 11.(a)The changes in the van der Waals volume (V w )(•), molecular volume (V M )(○), and void volume (V V )(▲) of various helical peptide and proteins as a function of molecular weight.The straight lines are the results of the least-squares analysis.The correlation coefficient values (R) of the least-square fit are 0.9736 for V w , 0.9769 for V M , and 0.9398 for V V , respectively.(b) Change in the V v per molecular weight of helical peptides and proteins as a function of molecular weight.
Fig. 12. FTIR (upper) and CD (lower) spectra of PLGA, BSA, and cytochrome c in water at 298 (solid line) and 77 K (long-dashed line).
The change in the intermolecular H-bond distance (d O ･･･ O ) between the NMA dimer's C=O group and the water molecule's O-D group is related to pressure-induced changes of the intermolecular H-bond distance in the -helical structure.We calculated the d O ･･･ O dependence of the amide I' frequencies of NMA dimer + D 2 O (Figure10).With decreasing d O ･･･ O (i.e., increasing pressure), the peak at 1632 cm  1 (•) shifts to lower frequency.Below d O ･･･ O = 2.36 Å, the frequency shift at 1632 cm -1 (•) increases significantly.Thus, with decreasing d O ･･･ O , the intermolecular H-bond between the NMA dimer's C=O group and the water molecule's O-D group strengthens.Because the peak at 1632 cm  1 shifts to a slightly extent than that at 1656 cm  1 , the average amide I' frequency shift for NMA dimer + D 2 O below d O ･･･ O = 2.36 Å is dominated by the peak at 1632 cm  1 .This lower amide I' frequency shift with decreasing d O ･･･ O (below d O ･･･ O = 2.36 Å) for NMA dimer + D 2 O is similar to that for the solvated -helical structure.Thus, our results indicate that pressure-induced shorting distance of the intermolecular H-bond between the peptide's C=O group and the water molecule's O-D group causes shifts of the amide I' mode to lower frequency for the solvated α-helical structure.Paschek et al., in a recent IR simulation study (Paschek et al., 2005), proposed that the pressure-induced shift of AK peptide's amide I' mode to lower frequency is due to hydration effects between the peptide and water molecules rather than structural changes in the peptide.Our results support this suggestion.The amide I' frequency shifts for the solvated -helical structure strongly correlate with changes in d C=O…D-O.

Table 1 .
Volume contributions (cm 3 /mol) of proteins by geometric volume calculation.