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Hydration of Lysozyme Studied by Raman Spectroscopy Vitaly Kocherbitov, Jekaterina Latynis, Audrius Misiunas, Justas Barauskas, and Gediminas Niaura J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp4017954 • Publication Date (Web): 04 Apr 2013 Downloaded from http://pubs.acs.org on April 16, 2013

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The Journal of Physical Chemistry

Hydration of Lysozyme Studied by Raman Spectroscopy Vitaly Kocherbitov, † Jekaterina Latynis,‡ Audrius Misiūnas,‡,§ Justas Barauskas,‡,† and Gediminas Niaura *,‡ †

Biomedical Science, Faculty of Health and Society, Malmö University, SE-20506 Malmö, Sweden;

‡

Vilnius University Institute of Biochemistry, Mokslininkų 12, LT-08662 Vilnius, Lithuania;

§

Institute of Chemistry, Center for Physical Sciences and Technology, Goštauto 9, LT-01108 Vilnius,

Lithuania; AUTHOR EMAIL ADDRESS [email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) CORRESPONDING AUTHOR FOOTNOTE Corresponding author: Gediminas Niaura. Phone: +370 52729642. Fax: +370 52729196. E-mail: [email protected]

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ABSTRACT

Hydration plays a fundamental role in maintaining the three dimensional structure and function of proteins. In this study Raman spectroscopy was used to probe the hydration induced structural changes at various sites of lysozyme at isothermal conditions in the range of water contents from 0 to 44 wt%. Raman hydration curves were constructed from detailed analysis of marker bands. Transition inflection points (wm) and onsets determined from the hydration curves have shown that structural changes start at 7−10 and end at about 35 wt% of water. The onset of structural changes coincides with the onset of the broad glass transition earlier observed in this system. The increase of α-helix content starts at very low concentrations of water with wm=12 wt%. Monitoring the development of important for the enzymatic action hydrophobic clusters has revealed the wm=15 wt% and completion of the process at 25 wt%. The parameters of 621 cm−1 (Phe) and 1448 cm−1 (CH2 bending) modes were found to be sensitive to hydration suggesting changes in organization of water molecules near the protein surface. The native structure of lysozyme was achieved at 35 wt% of water where its content is high enough for filling the space between lysozyme molecules.

Keywords: Glass transition, Marker bands, Protein, Hydrophobic cluster, Secondary structure, Tryptophan.


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Introduction Proteins in hydrated and dehydrated states have different properties. When dehydrated, proteins not only lose their biochemical function, but also undergo changes in dynamics and structure. A strong change in dynamics of proteins occurring during hydration/dehydration or temperature change is called the glass transition. The temperature of the glass transition (that occurs at given water content) is denoted as Tg. The glass transition is however a complex phenomenon and its transition temperature is dependent on the heating/cooling rate in calorimetric experiments or frequencies used in scattering or spectroscopic experiments.1 Moreover, some authors not only differentiate between the glass transition temperature Tg and the dynamic transition temperature, which is denoted as Td, but also claim that they have different mechanisms.2 Nonetheless, existence of glass transitions in proteins is well established and was studied using several techniques and methods. In general, fully hydrated proteins usually possess a broad glass transition at about 200K, whereas for partly dehydrated proteins the glass transition shifts to higher temperatures.3 Since water acts as a plasticizer, the glass transition can be monitored not only upon change of temperature, but also upon change of water content. Yang and Rupley4 studied the heat capacity of lysozyme-water mixture at 25 oC and found a strong increase of the heat capacity between 0.07 and 0.25 g/g (mass of water per mass of dry lysozyme). When compared with data obtained by other methods,3 this rise of the heat capacity can be attributed to a broad glass transition in the protein. A sorption calorimetric study performed at isothermal conditions at three temperatures also confirmed an existence of a glass transition in this concentration range.5 The fact that the glass transition in proteins is broader than in most glass-forming liquids and polymers might be explained by the heterogeneous structure of proteins. Indeed, in proteins there are many types of functional groups (charged, polar, non-polar) that have different energies of interactions with water and can have different dynamical properties. On the other hand, there is evidence that the glass transition in denatured lysozyme is much narrower than in native protein.6 This indicates that other factors than heterogeneity of protein functional groups is responsible for the broadness of the glass transitions in proteins. ACS Paragon Plus Environment

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Apart from the glass transition, the proteins undergo structural transition upon hydration and dehydration. The changes of protein structures as a function of hydration can be monitored using vibrational spectroscopy.7−19 For example, a FT-IR study of the amide III band showed that lysozyme has different secondary structures in the fully hydrated and in the lyophilized states.11 In particular, upon dehydration the content α-helixes decreases from 32% to 26%, while the amount of β-sheets increases from 18% to 44%. At the same time the amount of unordered material decreases upon dehydration. They also showed that these structural changes are essentially reversible. Griebenow and Klibanov10 also studied several proteins using the same approach and found the tendency that lyophlilization substantially increases the β-sheet content and lowers the α-helix content in all of them. The reason of the structural transitions in proteins is the necessity of the removal of the protein-air interface that may be formed upon dehydration.20 In the absence of water the protein molecules could not continuously fill the space without changing their structure. This would result in a drastic rise of the Gibbs energy of the system due to high surface tension. To prevent this, protein molecules adopt structures that can continuously fill the space.20 One important aspect of hydration-dehydration of proteins has not however been previously studied. Namely, the concentration range of the structural transition in proteins is still unknown because usually only extremes (either fully hydrated or dry lyophilized proteins) are investigated in vibrational spectroscopy studies. Moreover, the relationship between the dynamical and structural transitions in proteins has to the best of our knowledge not been studied. In this work we present a detailed Raman study of a partly hydrated lysozyme at a constant temperature in the range of water contents where both dynamical and structural transitions take place.


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Experimental Methods Materials. Lysozyme from chicken egg white (Cat. No. 62971, Lot# 0001356468) was purchased from Fluka Analytical (Sigma-Aldrich Co.). All other solvents and reagents were of analytical grade and were used as received. Milli-Q purified water was used for all experiments. Sample preparation. Before hydration procedures lysozyme was dried in a vacuum desiccator for at least 72 hours using CaCl2 or molecular sieves (type 3A) as sorbent. Hydration procedures of lysozyme were carried out as follows. Preparations with low concentration of water were prepared by spreading appropriate amounts (ca. 100 mg) of dried lysozyme on a large area of glass dish and incubating in a desiccator with a vapour atmosphere from saturated KCl solution (85.1 % relative humidity)21 for 10 – 1000 min at 20 °C. By using this procedure and depending on the incubation time lysozyme preparations containing up to 18 wt% of water were prepared. Preparations containing more than 18 wt% of water were prepared by weighing appropriate amounts (ca. 150 mg) of dried lysozyme and directly adding required amounts water. In all experiments the exact amount of absorbed water was determined by weighing lysozyme preparations before and after hydration procedure. Hydrated lysozyme preparations were immediately sealed and left to equilibrate at room temperature. In order to provide uniformity of water distribution in lysozyme particles of different sizes we equilibrated the samples at least for a weak after vapor adsorption. Bull22 demonstrated that the amount of water taken up by proteins does not change after 3−4 days of equilibration. It was later confirmed by other studies.23 Raman spectroscopy. Near-infrared Raman spectra were recorded using Echelle type spectrometer RamanFlex 400 (PerkinElmer, Inc.) equipped with thermoelectrically cooled (−50 °C) CCD camera and fiber-optic cable for excitation and collection of the Raman scattering. The 785 nm beam of the diode laser was used as the excitation source. The 180° scattering geometry was employed. The laser power at the sample was 100 mW and the beam was focused to a 200 µm diameter spot on the sample. The integration time was 10 s. Each spectrum was recorded by accumulation of 50 scans yielding overall integration time of 500 s. To increase signal-to-noise ratio 10 spectra were averaged. Samples were prepared in the cylindrical cells with quartz windows. The Raman frequencies were calibrated using the 5 ACS Paragon Plus Environment


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polystyrene standard (ASTM E 1840) spectrum. Intensities were calibrated by a NIST intensity standard (SRM 2241). All experiments were conducted at 20 °C. Raman spectra recorded at the beginning of experiment and after 3 h of continuous laser irradiation show no significant spectral changes indicating preservation of the sample integrity during the experiments (Figure S1, Supporting Information). Some experiments were performed with 632.8 nm excitation (He-Ne laser) by using the Raman microscope LabRam HR800 (Horiba Jobin Yvon) equipped with a grating containing 600 grooves/mm. Laser power at the sample was 10 mW. Raman spectra were taken using a 50x/0.50 NA long working distance objective or 10x/0.25 NA objective lens. For quantitative analysis of Raman bands the experimental contours were fitted by LorentzianGaussian or Gaussian form components using GRAMS A1 spectroscopy software (Thermo Fisher Scientific, Inc.) and assuming linear baseline.


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Results Recently, by using methods of sorption and desorption calorimetry the phase diagram of the lysozymewater system has been determined.5 According to the proposed model, the hydration of lysozyme occurs in four steps: (i) slow penetration of water into the protein-protein interface, (ii) gradual glass transition, (iii) further water uptake with disaggregation of protein aggregates, and (iv) accumulation of free water. The observed transitions are attributed to the changes in rigidity of the lysozyme molecule. Thus, at room temperature and up to 11 wt% of water lysozyme is in the rigid (glassy) state. In the water content region between 11 and 21 wt% lysozyme possess a glasslike dynamic transition from rigid to flexible (elastic) state due to the increase of mobility of the structural units. Above 21 wt% of water all lysozyme molecules are in the flexible state and above 35 wt% of water the mixture contains not only bound but also free water molecules. In order to fully understand the processes of hydration of lysozyme a combined characterization of the system by thermodynamic, dynamic and structural methods is however required. Although lysozyme is one of the most intensively studied proteins, majority of investigations are focused on energetics and dynamics of hydration, while structural aspects are seldom considered. Moreover, most of the structural studies refer only to extreme conditions, either fully hydrated or almost dry protein.10,11,24−29 Therefore, in this study we used Raman spectroscopy for detailed structural characterization of the whole hydration process of lysozyme in bulk conditions in the content range between 0 and 44 wt%. Overview of the Raman Spectrum of Lysozyme. Raman scattering is highly sensitive to the changes in secondary structure of proteins as well as to conformational changes of amino acid residues and its microenvironment, especially those possessing aromatic functional groups.30 Over the years numerous correlations between the parameters of Raman bands (wavenumber, intensity, and full width at half maximum, FWHM) and structure of amino acid residues and amide group have been established.30−35 In the present study a number of hydrated lysozyme preparations containing between 0 and 44 wt% of water were prepared and characterized by Raman spectroscopy. As an example, Figure 1 shows representative Raman spectra of completely dried lysozyme and preparation containing 37 wt% of water ACS Paragon Plus Environment

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in the wavenumber region of 600−1800 cm-1. Intensities are normalized to the intensity of well-defined Phe band at 1003 cm−1. The most intense bands in the spectra are associated with vibrations of aromatic amino acid functional groups and amide bond. It is known that lysozyme molecule contains 6 Trp, 3 Phe, 3 Tyr, and 1 His residues.36 Well-defined bands in the spectra correspond to vibrations of phenylalanine ring (621, 1003 and 1580 cm−1), tryptophan ring (758, 877, 1011, 1127, 1360, and 1552 cm-1), tyrosine ring (643, 851, and 1618 cm−1), and amide group (1253, 1336, and 1661 cm−1).30,35 A broad feature near 1447 cm−1 originates from the deformation motion of CH2 and CH3 groups, while band around 933 cm−1 corresponds to a backbone N−Cα−C stretching vibration of α-helix secondary structures. As seen from the Figure 1, hydration of lysozyme induces subtle changes in the parameters of several bands which are analyzed in more detail below.

Figure 1. A representative Raman spectra of lysozyme in the 600−1800 cm−1 wavenumber region at two hydration states (0 and 37 wt% of water). Excitation wavelength is 785 nm (100 mW) and integration time is 5000 s.


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Amide-I Band. Amide-I (Am-I) band primarily corresponds to C=O stretching vibration (83 %) with some contribution from out-of-phase C−N stretching, C−C−N deformation, and N−H in-plane bending vibrations.32,37 The position of Am-I mode depends on the strength of hydrogen bonding interaction (C=O•••H) involving the amide group and dipole-dipole interaction between the carboxyl groups. Weakening of hydrogen bonding interaction results in higher Am-I frequencies. The following wavenumber intervals were correlated with particular secondary structure elements:32,33,38−40 α-helix structure in the 1650−1660 cm-1 range, organized β-sheet structure in the 1670−1675 cm−1 window, and weakly hydrogen bonded or non-forming interstrand hydrogen bonds amide groups corresponding to loose β-strands, intermolecular β-sheet structures, polyproline II, and disordered structures which appear in the higher wavenumber spectral region of 1680−1695 cm−1. Figure 2A presents Raman spectra of lysozyme in Am-I spectral region at different states of hydration (0 and 37 wt% of water) and their difference spectrum. These spectra are similar to those reported earlier for dry and wet (0.47 g water/1 g of protein) lysozyme by using 514.5 nm laser excitation line.41 Figure 2A demonstrates that hydration of lysozyme shifts peak maximum to slightly lower wavenumbers indicating formation of increased amount of α-helix structures relative to other components.42 In addition, the intensity in the high frequency region decreases. Negative feature in the difference spectrum reveals hydration induced loss of the peak near 1687 cm−1. Such high frequency mode must originate from C=O groups which do not form hydrogen bonds or are involved in very weak C=O•••H interaction. Indeed, Am-I bands in the 1680−1695 cm−1 region have been associated with disordered structures, including β-turns, loose β-strands, and polyproline structures.38,39 We note that with increasing hydration level water bending mode near 1640 cm−1 may have some contribution to Am-I band. Analysis of water Raman spectrum and comparison with lysozyme sample at highest hydration level revealed negligible contribution of water band to 1687 cm−1 feature, however, some contribution to positive going band in the difference spectrum near 1635 cm−1 might take place (see Supporting Information). ACS Paragon Plus Environment

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Figure 2B shows the Raman difference intensities of the 1680−1695 cm−1 peak in Am-I region constructed by subtraction of the dried sample spectrum from that observed at certain hydration level intensity. For better observation the relative intensities are multiplied by −1. It is clear from Figure 2B that this negative-going band increases upon hydration indicating loss of the structures responsible for the 1687 cm-1 peak due to the formation of stronger hydrogen bonds. It should be noted that 1687 cm−1 peak of β-strands may be associated with intermolecular aggregation sites at low water content. Indeed, in the FT-IR studies of the secondary structure of proteins and peptides the high frequency component around 1675−1695 cm−1 has been reported to be a marker for aggregation of biomolecules through the formation of intermolecular β-sheet structures.33,40,43,44 Presented analysis suggests that with increasing water content this peak transforms to Raman component corresponding to α-helix or other secondary structure components possessing stronger hydrogen bonds.


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Figure 2. (A) Raman spectra of lysozyme in the Amide-I (Am-I) spectral region at two hydration states (0 and 37 wt% of water) and their difference spectrum. (B) The hydration curve corresponding to the Raman difference intensities 1680−1695 cm−1 peak in the difference spectra of Am-I region obtained by subtraction the spectrum recorded at 0 wt% of water from that at particular hydration. For better observation the relative intensities are multiplied by −1. Figure indicates that the peak near 1687 cm−1 in the spectrum of dry sample decreases upon hydration (transforms to Raman component corresponding to the formation of stronger hydrogen bonds). Solid line is the best fit to Equation 1 with the transition inflection point value wm of 15.7 ± 1.8 wt%.

The experimental data shown in Figure 2B were fitted by sigmoidal form curve assuming the twostate mechanism for the changes in studied vibrational mode with four parameters: ACS Paragon Plus Environment

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I = I0 +

a   w − wm   1 + exp −  b    

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(1)

where wm is the observed transition inflection point. The solid line presents the best fit to Equation 1 with wm = 15.7 ± 1.8 wt%. The wm values determined by the fitting procedure are listed in Table 1.

Table 1: Transition Inflection Points and Onsets Obtained from Raman Spectroscopic Analysis of Dependence of the Parameters of Spectral Bands on Water Content molecular

peak

group

frequency

assignment

Raman

transition

interpretation of hydration

marker

inflection

induced changes

point or (cm−1)

onset (wt% of water)

N−Cα−C

933

ν( N−Cα−C)

intensity

11.7 ± 1.6

increase in α-helix content

850/830

Tyr doublet,

intensity

13.7 ± 0.8

changes in H-bonding

Fermi resonance

ratio

between ring

I850/I830

main chain OH group of Tyr

interaction of Tyr

breathing mode Y1 and overtone of out-of-plane ring deformation mode 2 x Y16a Trp

1361

Fermi resonance

intensity

14.6 ± 1.6


increase in hydrophobicity of

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between in-plane

Trp ring environment,

ν(N−C) and

development of hydrophobic

combination

box

bands of ring out-of-plane deformations,W7 mode Trp

1552

ν(C2=C3), W3

FWHM

14.9 ± 1.8

conformation of indole rings around the C2=C3−Cβ−Cα

mode

moietya amide

1687

ν(C=O),

intensity

15.7 ± 1.8

sheet structures content

Am-I mode Trp

870

benzene ring

decrease of intermolecular β-

intensity

17.8 ± 1.3

formation of strong hydrogen

breathing +

bond of Trp-28 indole ring N1H

δ(N−H) ,W17

site

mode Met

695

ν(C−S)

intensity

20.3 ± 1.2

conformational changes in hydrophobic box due to movement of Met-105

Phe

621

in-plane ring deformation, F6b

intensity

onset near

hydrophobicity of the

12 wt%

environment at Phe sites;

mode

changes in water structure


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methylene

1448

δ(CH2),

intensity

scissoring mode

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onset near

changes in water structure near

12 wt%

the methylene groups and in dielectric properties of the environment

a

see Scheme 1 in Supporting Information for Trp ring structure and atom labeling. Abbreviations: ν,

stretching; δ, deformation


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N− −Cα α−C Stretching Vibration. The band in the vicinity of 933 cm−1 was used to monitor the content of α-helix in lysozyme at different hydration levels. It was earlier demonstrated that this mode is predominantly associated with the stretching vibration of N−Cα−C fragment and sensitively probes the presence of α-helix secondary structures in the Raman spectrum.30, 33, 45−47 Figure 3 shows the dependence of the intensity of Raman line near 933 cm−1 upon hydration. It should be noted that peak position shifts slightly to higher wavenumbers at higher water content (Figure 3A). The form of hydration curve is rather complex with maximum in the vicinity of 25−30 wt% of water content (Figure 3B). Interestingly, sharp increase in intensity of this band is visible even at low hydration levels. The obtained results indicate that development of α-helixes commence immediately after small amount of water was added to the system and stops after water content reached ~30 %. Further increase in water content resulted in slight decrease of α-helix structures. The transition inflection point for development of α-helix structures was found to be 11.7 ± 1.6 wt%.


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Figure 3. (A) Raman spectra of lysozyme in α-helix marker mode ν(N−Cα−C) spectral region at two hydration states (0 and 37 wt% of water). (B) Hydration induced dependence of the band intensity. Solid line is the best fit to Equation 1 with the transition inflection point value wm of 11.7 ± 1.6 wt%.

Tyr Fermi Doublet. Lysozyme molecule contains three tyrosine residues at sequence positions 20, 23, and 53.36 The Tyr-20 and Tyr-23 are positioned in the protein helical domain, while the Tyr-53 is situated in the beta domain.44 All Tyr residues are located around the active site of lysozyme.36 As shown in previous studies, Raman spectra of Tyr show intense doublet at 830/850 cm−1 arising from the Fermi resonance between the ring breathing mode (Y1) and overtone of an out-of-plane ring deformation (Y16a).31 The Fermi doublet sensitively probes the involvement of phenolic OH group in hydrogen bonding interaction.30,31, 48,49 Analysis of tyrosine model compounds in various solvents has revealed correlations as follows:31,48,49 (i) the highest intensity ratio I850/I830 of 6.7 is characteristic for


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the non-hydrogen bonded state of the phenolic group; (ii) the intensity ratio I850/I830 of 2.5 indicates that OH group acts as acceptor of strong hydrogen bond from an electropositive donor (for example −NH3+ group) and does not participate in hydrogen bonding via donation, (iii) the lower ratio value of 1.25 is characteristic for involvement of phenolic group in moderate both donor and acceptor H-bonding interaction (for example exposed to water molecules), and (iv) the low intensity ratio I850/I830 value of 0.3 indicates that OH group acts as a donor of strong H-bond (for example carboxyl oxygen). It is clear from Figure 4 that hydration induces drastic changes in hydrogen bonding interactions of tyrosine residues of lysozyme. At low water content the calculated Fermi doublet intensity ratio I850/I830 is approximately 1.5 (Figure 4B). With increasing water content it decreases reaching about 0.5 at the water content of 20 wt%. Similar value of 0.47 was observed for lysozyme in water solution.50 In the crystal state, only one of lysozyme tyrosine residue (Tyr-53) is involved in intramolecular hydrogen bonding.51 The relatively high ratio I850/I830 value in the initial dehydrated state indicates that phenolic group of at least one Tyr residue forms strong hydrogen bond with electropositive donor. With increasing hydration level water molecules first solvate charged residues.52 which might be responsible for weakening of interaction strength of Tyr residues with charged donors. Because Tyr residues are located near the active site of lysozyme, the hydration induced changes in hydrogen bonding interaction of phenolic groups might be important for recovery the physiological function of protein. The water content value corresponding to transition inflection point (wm) for Tyr doublet intensity ratio I850/I830 was found to be 13.7 ± 0.8 wt%. The transition was completely finished at approximately 20 wt% of water.


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Figure 4. (A) Representative Raman spectra of the hydration induced changes in Tyr doublet frequency region of lysozyme at 0, 16, and 37 wt% of water. Dotted lines represent bands deconvolution using Gaussian line shape components. Spectra are shifted vertically for clarity. (B) Dependence of the Tyr doublet Raman intensity ratio I850/I830 on level of hydration of lysozyme. Solid line is the best fit to Equation 1 with the transition inflection point value wm of 13.7 ± 0.8 wt%.

Trp Bands. Lysozyme has six tryptophan residues at sequence positions 28, 62, 63, 108, 111, and 123.36 Raman bands of tryptophan functional group are sensitive to conformation, hydrogen bonding interaction, and hydrophobicity of the environment surrounding the indole ring.30,34,53 It was demonstrated that tryptophan doublet, called W734 in the 1340−1373 cm−1 range, arising from the Fermi 18 ACS Paragon Plus Environment

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resonance between the fundamental in-plane N−C stretching mode and combination bands of the ring out-of-plane vibrations, sensitively probes the environment of the indole ring.54 In more hydrophobic environment the relative intensity of the mode near 1360 cm−1 increases when compared to the 1340 cm−1 component.34,50,51 As seen from the Figure 1, in our study we were not able to observe the lower frequency doublet component although the higher frequency band is clearly visible near 1360 cm−1. With increasing water content the intensity of this mode increases which indicates that indole rings of some Trp residues adopt more hydrophobic environment. Thus, presented data allow monitoring the development of hydrophobic clusters.17 The water content value corresponding to the transition inflection point for W7 mode was found to be 14.6 ± 1.3 wt%. The formation of hydrophobic clusters is completed at ~25 wt% of water.

Figure 5. Hydration induced dependence of the tryptophan W7 mode intensity in the Raman spectra of lysozyme. Solid line is the best fit to Equation 1 with the transition inflection point value wm of 14.6 ± 1.3 wt%.


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The second Trp band which displays clear changes upon hydration is W3 mode located near 1552 cm−1. This band arises mainly due to C2=C3 stretching vibration (see Scheme 1 in Supporting Information for Trp ring structure and atom labeling) contribution and serves as a conformation marker mode because of peak position sensitivity to the changes of torsional angle about the C2=C3−Cβ−Cα moiety.34 Our results show that position and intensity of the band remains intact while the bandwidth defined as a full width at half maximum (FWHM) clearly narrows at higher hydration level (Figure 6A). Such narrowing again indicates hydration induced changes in tertiary structure of the protein resulting in formation of hydrophobic pockets in the vicinity of Trp rings. Decrease in bandwidth is consistent with the view that more indole rings adopt similar conformation around the C2=C3−Cβ−Cα moiety. Analysis of hydration curve has revealed the transition inflection point value wm of 14.9 ± 1.8 wt%. This value is very similar to that found from the analysis of W7 mode and confirms hydration induced transformation of microenvironment near the indole rings.


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Figure 6. (A) Raman spectra of lysozyme in W3 spectral region at two hydration states. (B) Hydration induced dependence of the full width at half maximum (FWHM) of W3 mode. Solid line is the best fit to Equation 1 with the transition inflection point value wm of 14.9 ± 1.8 wt%.

The important Raman marker band for hydrogen bonding interaction of indole ring of Trp is the W17 mode located usually near 880 cm−1. This mode involves the deformation vibration of the N−H group of Trp and therefore is highly sensitive to the strength of the hydrogen bonding interaction at N1H site of indole ring.34,50,55,56 It was demonstrated that frequency of this band decreases with increasing strength of the H-bonding interaction.55 Raman spectrum in Figure 7A shows an averaged W17 peak of six Trp residues of lysozyme. In the spectrum of the dehydrated protein the peak position is near 877 cm−1. Upon addition of water (37 wt%) the band broadens and the shape becomes unsymmetrical. Observed


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peak frequency value corresponds to moderately hydrogen bonded indole ring.50 However, one can see that spectrum containing 37 wt% of water exhibits a shoulder (870 cm−1) near the intense band in the vicinity of 877 cm−1 which becomes clearly visible in the difference spectrum. Presence of this low frequency shoulder indicates that at least one Trp residue is involved in a very strong hydrogen bonding. Based on hydrogen-deuterium exchange kinetics Miura and co-workers have identified this residue as Trp-28.50 Constructed difference spectra allow probing the development of the intensity of strongly hydrogen bonded indole ring W17 band on the level of hydration (Figure 7B). Since the Trp-28 remains in hydrophobic box in solution phase,50,51 the transformation in tertiary structure of protein allowing formation of very strong hydrogen bond between the Trp-28 indole ring N1H group and carbonyl group of Tyr-23 takes place with increasing water content. The water content value corresponding to transition inflection point for low frequency W17 mode was found to be wm = 17.8 ± 1.3 wt%. The transformation process is completed at around 30 wt% of water.


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Figure 7. (A) Representative Raman spectra of the hydration induced changes in the tryptophan W17 mode spectral region at two hydration states (0 and 37 wt%) and their difference spectrum. (B) The hydration curve corresponding to the Raman difference intensities (870 cm−1 peak in the difference spectra of W17 region) obtained by subtraction the spectrum recorded at 0 wt% of water from that at particular water content. Solid line is the best fit to Equation 1 with the transition inflection point value wm of 17.8 ± 1.3 wt%.

C− −S Stretching Band. Lysozyme exhibits two intense bands in the spectral region where C−S stretching vibrations are expected to be observed (Figure 8). It is well known that C−S band frequency correlates with the structure of −S−CH2−CH2− group.57,58 Several rotational conformers denoted as ACS Paragon Plus Environment

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PC−T, PC−G, PH−T and PH−G have been identified, where the T and G symbols refer to trans and gauche conformations obtained by rotation around the −CH2−S− bond, while PH and PC indicate two possible rotational conformations obtained by rotation around the −CH2−CH2− group with the hydrogen and carbon atoms at the trans position with respect to the sulfur atom, respectively.57−60 Lysozyme contains eight Cys and two Met residues containing C−S bonds. The conformation of both Met residues (at 12 and 105 sequence positions) was found to be the same, i.e. PC−G.51 The band at 721 cm−1 belongs to PC−T conformer and has been attributed mainly to Cys residues.61 However, it should be noted that this mode may have some contribution from the S−C(H3) bond stretching vibration of Met residues. The 695-cm−1 peak can be assigned to C−S stretching vibration of PC−G conformer.59,60,62 Lord and Yu61 have attributed this peak to Met residues. As shown in Figure 8A, intensities of both bands increase with increasing hydration of lysozyme. In this work only the 695 cm−1 band has been analyzed in detail (Figure 8B). Intensification of this mode was observed during the binding of inhibitor to lysozyme.51 The Met-105 residue is situated in the hydrophobic box of protein between two indole rings (Trp-28 and Trp-111) which is adjacent to the active site. The changes in intensity of C−S stretching band of Met-105 were correlated with the moving of this residue from Trp-111 to Trp-28.51 Thus, increase in intensity of 695-cm−1 band may be associated with hydration induced conformational changes in hydrophobic box due to movement of Met-105 group. The hydration curve gives the inflection point wm of 20.3 ± 1.2 wt%. This value is rather high and indicates that rearrangement of Met residue takes place at the final stage of hydration induced conformational changes in lysozyme.


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Figure 8. (A) Raman spectra of lysozyme in C−S stretch spectral region at two hydration states. (B) Hydration induced dependence of the peak intensity of C−S stretching mode at 695 cm−1. Solid line is the best fit to Equation 1 with the transition inflection point value wm of 20.3 ± 1.2 wt%.

Deformation Mode of Phe Ring F6b. Phe ring exhibits characteristic for monoalkyl-substituted benzenes depolarized deformation band in the Raman spectrum near 621 cm−1 denoted as F6b.56 The strong and clear dependence of the intensity of F6b mode on the hydration level (Figure 9) suggests the effect of microenvironment near the Phe residues by water molecules. Lysozyme contains three Phe residues located on the surface of protein at positions of 3, 34, and 38.63 Thus, changes in spectroscopic parameters of this band must monitor the microenvironment near the Phe rings at the surface of lysozyme. Previously, it was shown that intensity of F6b band comparing with F12 one in lysozyme decreases on going from crystalline to aqueous solution phase.64 ACS Paragon Plus Environment

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Figure 9 B shows that intensity of F6b mode remains near constant until hydration reaches 13 wt%, which points on the association of water with other protein sites but not near the surface of Phe residues. Further increase in hydration results in near monotonic decrease in integrated intensity of the band, suggesting changes in microenvironment at the sites of Phe rings. In the initial dehydrated state lysozyme molecules are aggregated and Phe rings may be surrounded by the hydrophobic groups of the neighboring proteins. Decrease in intensity of F6b band most likely is associated with changes in hydrophobicity of microenvironment at Phe ring sites of the surface of protein due to penetration of water between the neighboring lysozyme molecules resulting in breaking the protein-protein contacts. Deep ultraviolet resonance Raman spectroscopic analysis revealed sensitivity of intensity of Phe bands to hydrophobicity of the environment because of influence of environment on the π-electron cloud of the substituted benzene ring.65,66 However, this conclusion might not be correct for non-resonant Raman spectra observed in this work. Therefore, to check this hypothesis we have conducted additional experiments with phenylalanine dissolved in water and water-acetonitrile mixture (1:1). The results are displayed in Figure S2 (Supporting Information). Small but clear decrease in relative intensity of F6b mode is visible on going from acetonitrile-water mixture to water. Presented data evidence the importance of solvation for decrease in relative intensity of F6b mode in Raman spectra excited with 785 nm. It should be noted that small upshift (0.7 cm−1) is visible for wavenumber of F12 mode in water solution. The plot of integrated peak intensity as a function of water content might be described by two straight lines. The point at which two lines intersect was found to be about 12 wt % of water. Such behavior of F6b peak intensity might be associated with abrupt changes in interaction of π-electron clouds of monosubstituted benzene rings with surrounding molecular groups. At higher hydration levels monotonic decrease in relative intensity of F6b mode might be associated with changes in organization of water molecules in the vicinity of Phe rings.


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Figure 9. (A) Raman spectra of lysozyme in F6b spectral region at two hydration states. (B) Hydration induced dependence of the integrated intensity of F6b mode.

Bending Vibration of CH2 Groups. Three bands are visible in the spectral region were bending vibration of methylene groups is expected to be observed (Figure 10). The higher frequency peak (1458 cm−1) is associated with deformation vibration of methyl groups, δ(CH3), while the peak near 1448 cm−1 corresponds to bending mode of CH2 groups. Intensities of both bands decrease at higher water content (Figure 10A). Parameters of the bands were determined by decomposition of the experimental contour into the three Lorentzian-Gaussian form components. The bending vibration of CH2 groups was analyzed in more details.


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Figure 10 clearly demonstrates that the intensity of CH2 bending band decreases upon hydration of lysozyme. However, the intensity dependence on hydration is not monotonic (Figure 10B); it remains near constant at low water content level (until 13 wt%) and sharply decreases at higher hydration values. The curve clearly contains the inflection point near 12 wt% of water content. Previous temperaturedependent Raman studies of crystalline lysozyme in the temperature interval from 77 to 273 K have revealed sudden decrease in relative intensity of δ(CH2) band in the region of 240−273 K.63 It was suggested that such temperature dependency might be associated with conformational changes around the CH2−CH2 bonds of some amino acid residues (glutamic acid and/or lysine) or changes in dynamic properties of proteins visible near 200 K.

Figure 10. (A) Raman spectra of lysozyme in methylene and methyl groups bending vibration spectral region at two hydration states (0 and 44 wt% of water). (B) Hydration induced dependence of the peak intensity of δ(CH2) mode at 1448 cm−1. ACS Paragon Plus Environment

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Discussion Understanding structural changes that occur in proteins upon hydration is a serious scientific challenge that requires combination of several experimental techniques. The advantage of Raman spectroscopy is in its unique possibility to monitor changes in structure and microenvironment of particular molecular groups during the hydration process. Table 1 and Figures 2−10 indicate what changes in tertiary and secondary structure of lysozyme take place upon hydration of protein as viewed by Raman spectroscopy. Below we combine our results with the data obtained by other methods to describe the main steps of hydration of lysozyme from the dry state to fully hydrated protein. In completely dry state the lysozyme is different from the native form both structurally and dynamically. From dynamical point of view it is in the glassy state and from structural point of view the content of β-structures is higher while the content of α-helixes is lower than in the native state. In the glassy state which according to calorimetric data4,5 lasts up to 7-8 wt% no significant changes in the structure of lysozyme are seen upon hydration at 25 oC. This can be explained by the fact that in the glassy state all changes are very slow and even when they exist they cannot be monitored during experiment time. In the beginning of the broad glass transition that lasts from 7 to about 20 wt%, structural changes start to occur. The values of the transition inflection points shown in Table 1 are different for different groups; nonetheless according to Figures 2−10 most of transitions gradually start at about 7−10 wt% of water, i.e. in the beginning of the glass transition. The lowest inflection point at 11.7 wt% has the α-helix marker band. α-Helixes dominate the structure of native lysozyme and being rather stable structures they start to form first in the gradual transition from dry to hydrated lysozyme. Transition that corresponds to hydration-induced destruction of intermolecular β-sheets is stretched over a broader concentration range and its inflection point is at higher water content of 15.7 wt% (Figure 2A). Interestingly, it coincides with the value of the Langmuir monolayer coverage of the interface between lysozyme molecules (15.5 wt%) determined using water sorption calorimetry.20 This shows that


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the two processes – adsorption of water and formation of β-sheets compete in this concentration range. This implies that intermolecular β-sheets which exist in the interface between dry neighboring lysozyme molecules are disrupted when water molecules are absorbed by the protein. The threshold of the dielectric response in lysozyme powder at h=0.152 (water to dry protein mass ratio) or 13 wt% of water67 is just below the water monolayer coverage concentration. This threshold is attributed to proton surface percolation transition, in other words to appearance of long-range connectivity between water molecules67. Understandably, the water content where this happens is slightly lower than the monolayer coverage concentration. A related effect is observed during the analysis of the methylene and methyl groups bending vibration spectra and also in deformation mode of Phe (see Figures 9 and 10). Up to 12−13% of water the intensities do not change much but at higher concentrations a clear decrease is observed. Unlike behavior of other Raman markers considered in this work, these intensities do not come to plateau values at higher water contents. This can be explained by the fact that neither methylene nor Phe ring are directly involved in hydrogen bonding. They therefore probe not particular structural changes induced by hydrogen bonding (such changes normally should have sigmoidal shapes with plateaus in the beginning and at the end); but the general dielectric environment of the system that continues to change even at relatively high water contents. Analysis of data presented in Figures 2, 5, 6, 7 and 8 suggests that changes in lysozyme structure last up to 30−40 wt% which indicates that the native tertiary structure is not fully formed until these values of water content are reached. The lowest amount of water needed for formation of the native structure of the protein can be estimated from simple geometrical considerations. A lysozyme molecule can be schematically represented by a prolate spheroid with long to short axis ratio 5:3. A biopolymer-water system can in ambient conditions exist only if it continuously fills the space.20 Any significant discontinuities in the space filling would result in enormous increase of the Gibbs energy of the system due to surface tension. Therefore, the amount of water needed to fill the space between randomly packed prolate spheroids would be the lowest theoretical limit for existence of native structure in an amorphous protein. According to simulations by Donev et al.68 the volume fraction of packed prolate spheroids with 30 ACS Paragon Plus Environment

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aspect ratio 5:3 is about 0.715. In other words, minimum theoretical water content needed for existence of native structure in lysozyme is 28.5 vol% or 22 wt% assuming density of lysozyme of 1.4 g/cm3.69 In reality, the minimum water content should be higher because we expect at least one layer of water molecules being present at the interface between lysozyme molecules. This leads to the following ratio:

VW 0.285 = , VL + VBW 0.715

(2)

where VW , VL and VBW are volumes of free water, lysozyme and bound water respectively. Calculations of the area and the volume of a prolate spheroid based on approximate dimensions of lysozyme molecules of 5x3 nm give 4150 Å2 and 23560 Å3 respectively. Assuming that a half of a water molecule belongs to every lysozyme molecule at the “touching” interface, the volume of the bound water is 6225 Å3 which gives the volume of free water of 11870 Å3. Finally, the water to lysozyme ratio is calculated as

h=

VW + VBW d LVL

(3)

Using density of native lysozyme dL of about 1.4 g/cm3,68 one arrives at the h value of 0.55 or 35 wt% of water. Thus, from simple geometrical considerations, at about 35 wt% of water one can expect formation of native structure of lysozyme. This value is in agreement with data presented in Figures 2, 5, 6, 7 and 8. Moreover, a desorption calorimetric data5 show that at 35 wt% a very pronounced step in the activity of water and in partial molar enthalpy of mixing of water is observed upon removal of water from lysozyme solution. Thus, at water contents above 35 wt% dehydration does not change the lysozyme structure and only removal of water occurs. Below 35 wt% of water dehydration has an effect on the structure of lysozyme because further removal of water cannot happen without geometrical distortion of lysozyme molecules. ACS Paragon Plus Environment

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Conclusions We have presented a detailed analysis of hydration-induced changes in parameters of Raman marker bands of aromatic amino acid side chains and bands sensitive to secondary structure of lysozyme. In addition, the behavior of Phe ring F6b vibration, C−S stretching, and CH2 bending modes were probed by Raman spectroscopy using near-infrared excitation at 785 nm in the range of water contents from 0 to 44 wt%. Raman spectroscopy was able to provide insight into the hydration-induced structural changes at various sites of lysozyme molecule. Dependence of parameters of Raman bands on water content (Raman hydration curves) revealed that structural changes in the protein start at ~ 7−10 wt% and end at ~ 35 wt% of hydration level. The onset of the structural changes observed by Raman spectroscopy was found to be close to onset of broad glass transition detected earlier in this system. Analysis of N−Cα−C stretching band revealed folding of α−helixes with transition inflection point near 12 wt% of water content, while considerably higher hydration level (inflection point at ~ 16 wt%) was required for water-induced destruction of intermolecular β−sheets between the neighboring peptides. Development of hydrophobic box required for the enzymatic function of lysozyme was monitored by intensity changes of Trp W7 mode at 1360 cm−1. The inflection point for this process in Raman hydration curve was found to be near 15 wt%. Similar hydration value was characteristic for rotation conformational changes of Trp side chains obtained from the analysis of changes in the full width at half maximum of the Trp W3 mode at 1552 cm−1. Intensification of C−S stretching bands at higher water content was correlated with the movement of Met-105 residue in the hydrophobic box. Formation of hydrophobic clusters was completed at ~ 25 wt%. The intensity ratio of Tyr doublet (I850/I830) decreased from 1.5 to 0.5 upon increasing water content with transition inflection point near 14 wt% indicating weakening of interaction strength of Tyr phenolic group with charged residues in protein upon hydration. Because Tyr residues are located near the active site of lysozyme, the hydration induced transformations in hydrogen bonding interaction might be


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important for recovery the physiological function of enzyme. The transition associated with Tyr residues was completely finished at ~ 20 wt%. Near linear decrease in intensities of Phe ring F6b (621 cm−1) mode and CH2 bending vibration of aliphatic side chains (1448 cm−1) was observed at hydration level higher than 12 wt%. These changes were explained as associated with continuous changes in structure of water network near the protein surface. In general, spectroscopic data revealed that lysozyme adopts its native structure at 35 wt% of water content. Such amount of water is sufficient for filling the space between the protein molecules.

Acknowledgement. This research was funded by a grant (No. MIP-129/2010) from the Research Council of Lithuania and the Swedish Institute Baltic Sea Region Exchange Program (VISBY) (No. 00885/2009). Jekaterina Latynis acknowledges Student Research Fellowship Award from the Lithuanian Science Council.

Supporting Information Available Analysis of laser radiation effect on the sample integrity, analysis of water bending mode effect on parameters of Amide-I band, Trp ring structure and atom labeling scheme, and Raman spectra of phenylalanine dissolved in water and water : acetonitrile mixture. This information is available free of charge via the Internet at http://pubs.acs.org


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REFERENCES 1.

Donth, E. The Glass Transition; Springer-Verlag: Berlin, Germany, 2001.

2.

Khodadadi, S.; Malkovskiy, A.; Kisliuk, A.; Sokolov, A. P. A Broad Glass Transition in Hydrated Proteins. Biochim. Biophys. Acta Protein Proteonomics 2010, 1804, 15−19.

3.

Gregory, R. B. Protein-Solvent Interactions; Marcel Dekker: New York, New York, 1994.

4.

Yang, P.–H.; Rupley, J. A. Protein-Water Interactions. Heat Capacity of the Lysozyme-Water System. Biochemistry 1979, 18, 2654−2661.

5.

Kocherbitov, V.; Arnebrant, T.; Söderman, O. Lysozyme−Water Interactions Studied by Sorption Calorimetry. J. Phys. Chem. B 2004, 108, 19036−19042.

6.

Kocherbitov, V.; Arnebrant, T. Hydration of Thermally Denaturated Lysozyme Studied by Sorption Calorimetry and Differential Scanning Calorimetry. J. Phys. Chem. B 2006, 110, 10144−10150.

7.

Poole, P. L.; Finey, J. L. Sequential Hydration of Dry Proteins: A Direct Difference IR Investigation of Sequence Homologs Lysozyme and α-lactalbumin. Biopolymers 1984, 23, 1647−1666.

8.

Poole, P.L.; Finey, J.L. Hydration-Induced Conformational and Flexibility Changes in Lysozyme at Low Water Content. Int. J. Biol. Macromol. 1983, 5, 308−310.

9.

Poole, P.L. The Role of Hydration in Lysozyme Structure and Activity – Relevance in Protein Engineering and Design. J. Food Eng. 1994, 22, 349−365.


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Page 35 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10. Griebenow, K.; Klibanov, A. M. Proc. Lyophilization-Induced Reversible Changes in the Secondary Structure of Proteins. Natl. Acad. Sci. U.S.A. 1995, 92, 10969−10976. 11. Costantino, H. R.; Griebenow, K.; Mishra, P.; Langer, R.; Klibanov, A. M. Fourier-Transform Infrared Spectroscopic Investigation of Protein Stability in the Lyophilized Form. Biochim. Biophys. Acta: Protein Structure Mol. Enzymol. 1995, 1253, 69−74. 12. Mallamace, F.; Chen, S. H.; Broccio, M.; Corsaro, C.; Crupi, V.; Majolino, D.; Venuti, V.; Baglioni, P.; Fratini, E.; Vannucci, C.; Stanley, H. E. Role of the Solvent in the Dynamical Transitions of Proteins: The Case of the Lysozyme-Water System. J. Chem. Phys. 2007, 127, 045104. 13. Georget, D.M.R.; Belton, P.S. Effects of Temperature and Water Content on the Secondary Structure of Wheat Gluten Studied by FTIR Spectroscopy. Biomacromolecules 2006, 7, 469−475. 14. Liltorp, K.; Maréchal, Y. Hydration of Lysozyme as Observed by Infrared Spectrometry. Biopolymers 2005, 79, 185−196. 15. Careri, G.; Giansanti, A.; Gratton, E. Lysozyme Film Hydration Events. An IR and Gravimetric Study. Biopolymers 1979, 18, 1187−1203. 16. Rupley, J.A.; Gratton, E.; Careri, G. Water and Globular Proteins. Trends Biochem. Sci. 1983, 8, 18−22. 17. Shou, J.-J.; Zeng, G.; Zhang, Y.-H.; Lu (Max), G.Q. Micro-Raman Spectroscopic Observation of Water Expulsion Induced Destruction of Hydrophobic Clusters in Crystalline Lysozyme. J. Phys. Chem. B 2009, 113, 9633−9635.


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Page 36 of 53

18. Mukherjee, S.; Chowdhury, P.; Gai, F. Infrared Study of the Effect of Hydration on the Amide I Band and Aggregation Properties of Helical Peptides. J. Phys. Chem. B 2007, 111, 4596−4602. 19. Dong, J.; Hubel, A.; Bischof, J.C.; Aksan, A. Freezing-Induced Phase Separation and Spatial Microheterogeneity in Protein Solutions. J. Phys. Chem. B 2009, 113, 10081−10087. 20. Kocherbitov, V.; Arnebrant, T. Hydration of Lysozyme: The Protein-Protein Interface and the Enthalpy-Entropy Compensation. Langmuir 2010, 26, 3918−3922. 21. Greenspan, L. Humidity Fixed Points of Binary Saturated Aqueous Solutions. J. Res. Natl. Bur. Stand. 1977, 81A, 89−96. 22. Bull, H.B. Adsorption of Water Vapor by Proteins. J. Am. Chem. Soc. 1944, 66, 1499−1507. 23. Hageman, M. J.; Possert, P. L.; Bauer, J. M.; Prediction and Characterization of the Water Sorption Isotherm for Bovine Somatotropin. J. Agric. Food Chem. 1992, 40, 342−347. 24. Luthra, S.; Kalonia, D. S.; Pikal, M. J. Effect of Hydration on the Secondary Structure of Lyophilized Proteins as Measured by Fourier Transform Wrared (FTIR) Spectroscopy. J. Pharm. Sci. 2007, 96, 2910−2921. 25. Sinha, S.; Li, Y.; Williams, T. D.; Topp, E. M. Protein Conformation in Amorphous Solids by FTIR and by Hydrogen/Deuterium Exchange with Mass Spectrometry. Biophys. J. 2008, 95, 5951−5961. 26. Hédoux, A.; Ionov, R.; Willart, J.-F.; Lerbret, A.; Affouard, F.; Guinet, Y.; Descamps, M.; Prévost, D.; Paccou, L.; Danéde, F. Evidence of a Two-Stage Thermal Denaturation Process in Lysozyme: A Raman Scattering and Differential Scanning Calorimetry Investigation. J. Chem. Phys. 2006, 124, 014703.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


27. Hédoux, A.; Guinet, Y.; Paccou, L. Analysis of the Mechanism of Lysozyme Pressure Denaturation from Raman Spectroscopy Investigations, and Comparison with Thermal Denaturation. J. Phys. Chem. B 2011, 115, 6740−6748. 28. Zhao, W.; Yang, R. Experimental Study on Conformational Changes of Lysozyme in Solution Induced by Pulsed Electric Field and Thermal Stress. J. Phys. Chem. B 2010, 114, 503−510. 29. Torreggiani, A.; Foggia, M. Di.; Manco, I.; Maio, A. De.; Markarian, S.A.; Bonora, S. Effect of Sulfoxides on the Thermal Denaturation of Hen Lysozyme: A Calorimetric and Raman Study. J. Mol. Struct. 2008, 891, 115−122. 30. Wen, Z. -Q. Raman Spectroscopy of Protein Pharmaceuticals. J. Pharm. Sci. 2007, 96, 2861−2878. 31. Siamwiza, M. N.; Lord, R. C.; Chen, M. C.; Takamatsu-Harada, I; Matsuura, H; Shimanouchi, T. Interpretation of the Doublet at 850 and 830 cm−1 in the Raman Spectra of Tyrosyl Residues in Proteins and Certain Model Compounds. Biochemistry 1975, 14, 4870−4876. 32. Krimm, S.; Bandekar, J. Vibrational Spectroscopy and Conformation of Peptides, Polypeptides, and Proteins. Adv. Protein Chem. 1986, 38, 181−364. 33. Jackson, M.; Mantsch, H. H. The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure. Crit. Rev. Biochem. Mol. Biol. 1995, 30, 95−120. 34. Takeuchi, H. Raman Structural Markers of Tryptophan and Histidine Side Chains in Proteins. Biopolymers 2003, 72, 305−317. 35. Niaura, G. Raman Spectroscopy in Analysis of Biomolecules. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley & Sons Ltd.: Chichester, UK, 2008; pp 1−34.


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Page 38 of 53

36. Blake, C.C.F.; Koenig, D.F.; Mair, G.A.; North, A.C.T.; Phillips, D.C.; Sarma, V.R. Structure of Hen Egg-white Lysozyme : A Three-Dimensional Fourier Synthesis at 2 Å Resolution. Nature

1965, 4986, 757−761. 37. Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Determination of protein Secondary Structure by Fourier-Transform Infrared Spectroscopy – A Critical Assessment. Biochemistry 1993, 32, 389−394. 38. Carey, P. R. Raman Crystallography and Other Biochemical Applications of Raman Microscopy. Annu. Rev. Phys. Chem. 2006, 57, 527−554. 39. Huang, K.; Maiti, N. C.; Phillips, N. B.; Carey, P. R.; Weiss, M. A. Structure-Specific Effects of Protein Topology on Cross-β Assembly: Studies of Insulin Fibrillation. Biochemistry 2006, 45, 10278−10293. 40. Wolkers, W.F.; Van Kilsdonk M.G.; Hoekstra, F.A. Dehydration-Induced Conformational Changes of Poly-L-lysine as Influenced by Drying Rate and Carbohydrates. Biochim. Biophys. Acta 1998, 1425, 127−136. 41. Caliskan, G.; Mechtani, D.; Roh, J.H.; Kisliuk, A.; Sokolov, A.P.; Azzam, S.; Cicerone, M.T.; Lin-Gibson, S.; Peral, I. Protein and Solvent Dynamics: How Strongly Are They Coupled? J. Chem. Phys. 2004, 121, 1978−1983. 42. Torreggiani, A.; Tamba, M.; Manco, I.; Faraone-Mennella, M.R.; Ferreri, C.; Chatgilialoglu, C. Radiation Damage of Lysozyme in a Biomimetic Model: Some Insights by Raman Spectroscopy. J. Mol. Struct. 2005, 744-747, 767−773. 43. Dong, A.; Randolph, T.W.; Carpenter, J.F. Entrapping Intermediates of Thermal Aggregation in α-Helical Proteins with Low Concentration of Guanidine Hydrochloride. J. Biol. Chem. 2000, 275, 27689−27693. ACS Paragon Plus Environment

38

Page 39 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


44. Meersman, F.; Heremans, K. Temperature-Induced Dissociation of Protein Aggregates: Accessing the Denatured State. Biochemistry 2003, 42, 14234−14241. 45. Blanpain-Avet, P.; Hédoux, A. ; Guinet, Y. ; Paccou, L. ; Petit, J. ; Six, T. ; Delaplace, G. Analysis by Raman Spectroscopy of the Conformational Structure of Whey Proteins Constituting Fouling Deposits During the Processing in a Heat Exchanger. J. Food Engineer. 2012, 110, 86−94. 46. Tu, A.T. Peptide Backbone Conformation and Microenvironment of Protein Side Chains. In Spectroscopy of Biological Systems, Advances I Spectroscopy (Clark, R.J.H., and Hester, R.E., Eds) 1986, Vol. 13, pp. 47−111, Wiley, Chichester. 47. Ford, S. J.; Cooper, A.; Hecht, L.; Wilson, G.; Barron, L. D. Vibrational Raman Optical Activity of Lysozyme – Hydrogen-Deuterium Exchange, Unfolding and Ligand Binding. J. Chem. Soc. Faraday Trans. 1995, 91, 2087−2093. 48. Arp, Z.; Autrey, D.; Laane, J.; Overman, S.A.; Thomas, G.J., Jr. Structural Studies of Viruses by Raman Spectroscopy Part LXXI – Tyrosine Raman Signatures of the Filamentous Virus Ff are Diagnostic of Non-hydrogen-bonded Phenoxyls: Demonstration by Raman and Infrared Spectroscopy of p-Cresol Vapor. Biochemistry 2001, 40, 2522−2529. 49. Overman, S.A.; Aubrey, K.L.; Vispo, N.S.; Cesareni, G.; Thomas, G.J., Jr. Novel Tyrosine Markers in Raman Spectra of Wild-Type and Mutant (Y21M and Y24M) Ff Virions Indicate Unusual Environments for Coat Protein Phenoxyls. 46. Studies of Virus Structure by Laser Raman Spectroscopy. Biochemistry 1994, 33, 1037−1042. 50. Miura, T.; Takeuchi, H.; Harada, I. Characterization of Individual Tryptophan Side Chains in Proteins Using Raman Spectroscopy and Hydrogen-Deuterium Exchange Kinetics. Biochemistry

1988, 27, 88−94. ACS Paragon Plus Environment

39


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 53

51. Miura, T.; Takeuchi, H.; Harada, I. Raman Spectroscopic Characterization of Tryptophan Side Chains in Lysozyme Bound to Inhibitors: Role of the Hydrophobic Box in the Enzymatic Function. Biochemistry 1991, 30, 6074−6080. 52. Zhou, H.-X. A Unified Picture of Protein Hydration: Prediction of Hydrodynamic Properties from Known Structures. Biophys. Chem. 2001, 93, 171−179. 53. Misiunas, A.; Talaikyte, Z.; Niaura, G.; Razumas, V.; Nylander, T. Thermomyces Lanuginosus Lipase in the Liquid-Crystalline Phases of Aqueous Phytantriol: X-ray Diffraction and Vibrational Spectroscopic Studies. Biophys. Chem. 2008, 134, 144−156. 54. Harada, I.; Miura, T.; Takeuchi, H. Origin of the Doublet at 1360 and 1340 cm−1 in the Raman Spectra of Tryptophan and Related Compounds. Spectrochim. Acta A 1986, 42, 307−312. 55. Takeuchi, H.; Harada, I. Normal Coordinate Analysis of the Indole Ring. Spectrochim. Acta A

1986, 42, 1069−1078. 56. Harada, I.; Takeuchi, H. Raman and Ultraviolet Resonance Raman Spectra of Proteins and Related Compounds. In Spectroscopy of Biological Systems, Advances I Spectroscopy (Clark, R.J.H., and Hester, R.E., Eds) 1986, Vol. 13, pp. 113−175, Wiley, Chichester. 57. Nogami, N.; Sugeta, H.; Miyazawa, H. C−S Stretching Vibrations and Molecular Conformations of Isobutyl Methyl Sulfide and Related Alkyl Sulfides. Bull. Chem. Soc. Jpn. 1975, 48, 2417−2420. 58. Li, H.; Wurrey, C.J.; Thomas, G.J., Jr. Structural Studies of Viruses by Laser Raman Spectroscopy. 36. Cysteine Conformation and Sulfhydryl Interactions in Proteins and Viruses. 2. Normal Coordinate Analysis of the Cysteine Side Chain in Model Compounds. J. Am. Chem. Soc. 1992, 114, 7463−7469.


40

Page 41 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


59. Podstawka, E.; Ozaki, Y.; Proniewicz, L.M. Part II: Surface-Enhanced Raman Spectroscopy Investigation of Methionine Containing Heterodipeptides Adsorbed on Colloidal Silver. Appl. Spectrosc. 2004, 58, 581−590. 60. Podstawka, E.; Azaki, Y.; Proniewicz, L.M. Adsorption of S−S Containing Proteins on a Colloidal Silver Surface Studied by Surface-Enhanced Raman Spectroscopy. Appl. Spectrosc.

2004, 58, 1147−1156. 61. Lord, R.C.; Yu, N.-T. Laser-Excited Raman Spectroscopy of Biomolecules. J. Mol. Biol. 1970, 50, 509−524. 62. Lee, H.I.; Kim, M.S.; Suh, S.W. A Raman Spectroscopic Study on Subtilisin Family. Korean Biochem. J. 1989, 22, 321−326. 63. Nara, M.; Sakamoto, A.; Yamamichi, J.; Tasumi, M. Temperature Dependence of Near-IR Excited Raman Spectra of Crystalline Hen Egg-White Lysozyme. Biopolymers 2001, 62, 168−172. 64. Yu, N.-T.; Jo, B.H. Comparison of Protein Structure in Crystals and in Solution by Laser Raman Scattering. Arch. Biochem. Biophys. 1973, 156, 469−474. 65. Hildebrandt, P.G.; Copeland, R.A.; Spiro, T.G.; Otlewski, J.; Laskowski, M., Jr.; Prendergast, F.G. Tyrosine Hydrogen-Bonding and Environmental Effects in Proteins Probed by Ultraviolet Resonance Raman Spectroscopy. Biochemistry 1988, 27, 5426−5433. 66. Xu, M.; Ermolenkov, V.V.; Uversky, V.N.; Lednev, I.K. Hen Egg White Lysozyme Fibrillation: a Deep-UV Resonance Raman Spectroscopic Study. J. Biophoton. 2008, 1, 215−229. 67. Careri, G.; Giansanti, A.; Rupley, J.A. Proton Percolation on Hydrated Lysozyme Powders. Proc. Natl Acad. Sci. USA. 1986, 83, 6810−6814. ACS Paragon Plus Environment

41


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 53

68. Donev, A.; Cisse, I.; Sachs, D.; Variano, E.; Stillinger, F.H.; Connelly, R.; Torquato, S.; Chaikin, P.M. Improving the Density of Jammed Disordered Packings Using Ellipsoids. Science 2004, 303, 990−993. 69. Fischer, H.; Polikarpov, I.; Craievich, A.F. Average Protein Density is a Molecular-WeightDependent Function. Protein Science 2004, 13, 2825−2828.


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Hydration of Lysozyme Studied by Raman Spectroscopy - The Journal

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