J. Phys. Chem. B 2009, 113, 14769–14778
14769
Hydrogen Bonding and Solvent Polarity Markers in the UV Resonance Raman Spectrum of Tryptophan: Application to Membrane Proteins Diana E. Schlamadinger, Jonathan E. Gable, and Judy E. Kim* Department of Chemistry and Biochemistry, UniVersity of California at San Diego, 9500 Gilman DriVe, La Jolla, California 92093 ReceiVed: June 10, 2009; ReVised Manuscript ReceiVed: September 5, 2009
Ultraviolet resonance Raman (UVRR) spectra of tryptophan compounds in various solvents and a model peptide are presented and reveal systematic changes that reflect solvent polarity, hydrogen bond strength, and cation-π interaction. The commonly utilized UVRR spectral marker for environment polarity that has been based on off-resonance Raman data, the tryptophan Fermi doublet ratio I∼1360/I∼1340, exhibits different values in on- and off-resonance Raman spectra as well as for different tryptophan derivatives. Specifically, the UVRR Fermi doublet ratio for indole ranges from 0.3 in polar solvents to 0.8 in nonpolar solvents, whereas the respective values reported here and previously for off-resonance Raman spectra are 0.5-1.3. UVRR Fermi doublet ratios for the more biologically relevant molecule, N-acetyl tryptophan ethyl ester (NATEE), are in a smaller range of 1.1 (polar solvent) to 1.7 (nonpolar solvent) and correlate to the solvent polarity/polarization parameters π* and ENT . As has been reported previously, several UVRR modes are also sensitive to the hydrogen bond strength of the indole N-H moiety. Here, we report a new unambiguous marker for H-bonding: the ratio of the W10 (∼1237 cm-1) intensity to that of the W9 (∼1254 cm-1) mode (RW10). This ratio is 0.7 for NATEE in the absence of hydrogen bond acceptors and increases to 3.1 in the presence of strong hydrogen bond acceptors, with a value of 2.3 in water. The W8 and W17 modes shift more than +10 and approximately -5 cm-1 upon increase in hydrogen bond strength; this range for W17 is smaller than that reported previously and reflects a more realistic range for proteins and peptides in solution. Finally, our data provide evidence for change in the W18 and W16 relative intensity in the presence of cation-π interactions. These UVRR markers are utilized to interpret spectra of model membrane-bound systems tryptophan octyl ester and the peptide toxin melittin. These spectra reveal the importance of intra- and intermolecular hydrogen bonding and cation-π interactions that likely influence the partitioning of membrane-associated biomolecules to lipid bilayers or self-associated soluble oligomers. The UVRR analysis presented here modifies and augments prior reports and provides an unambiguous set of spectral makers that can be applied to elucidate the molecular microenvironment and structure of a wide range of complex systems, including anchoring tryptophan residues in membrane proteins and peptides. Introduction Tryptophan is the least abundant residue in soluble proteins, accounting for only 1.1% of the amino acids expressed in cytoplasmic proteins,1 but is more prevalent in membrane proteins, with an abundance of 2.9% in transmembrane R-helical domains.2 This aromatic residue typically plays key functional roles in proteins because of its unique properties among the 20 natural amino acids: tryptophan exhibits the largest accessible nonpolar surface area that is highly polarizable, possesses an indole N-H moiety that is capable of hydrogen bond donation, and displays the greatest electrostatic potential for cation-π interactions.3,4 These important physical properties render tryptophan an ideal amphiphilic residue with the greatest propensity to reside in the interfacial region of a membrane protein as compared to any other naturally occurring amino acid.5 Tryptophan has been found to stabilize membrane spanning proteins and peptides by acting as anchors along the interface of the bilayer.6,7 For example, replacement of tryptophan residues with phenylalanine in the 325-residue integral membrane protein, outer membrane protein A, destabilizes the protein * Corresponding author. Phone: 858-534-8080. Fax: 858-534-7042. E-mail:
[email protected].
relative to wild-type when folded into lipid bilayers.8,9 Tryptophan residues in membrane-associated antimicrobial peptides also play important functional roles in hemolytic and bactericidal activity.10,11 In the antibiotic channel peptide gramicidin A, substitution of tryptophan for phenylalanine residues results in reduction of antibacterial activity.12,13 These and other examples illustrate that the presence, location, and environment of tryptophan residues are critical in the study of folding and insertion of membrane proteins and membrane-associated peptides. Tryptophan displays environment-sensitive photophysical properties. Tryptophan fluorescence has been widely utilized to reveal its local environment in proteins, accessibility to solvent, and location with respect to another biological (e.g., heme) or exogenous (fluorescence acceptor) moiety. UV resonance Raman (UVRR) spectroscopy is a vibrational technique that selectively enhances signal from absorbing chromophores and has also been applied to the study of tryptophan and proteins. One of the biggest advantages of UVRR over fluorescence and other electronic spectroscopy techniques is that UVRR inherently reports on environment and structure, including hydrogen bonding states and orientation with respect to backbone, with great selectivity and sensitivity.14–16 UVRR may
10.1021/jp905473y CCC: $40.75 2009 American Chemical Society Published on Web 10/09/2009
14770
J. Phys. Chem. B, Vol. 113, No. 44, 2009
also be applied to probe different protein moieties, including other residues, such as tyrosine and proline, as well as the amide backbone to obtain a more comprehensive picture of the biomolecule. The commercial availability of tunable deep-UV lasers has resulted in a vast number of UVRR studies of tryptophan residues in proteins over the past two decades.17–20 However, interpretation of these data has generally relied on data of L-tryptophan and model tryptophan derivatives that were acquired under off-resonance conditions.21–23 Detailed UVRR studies on tryptophan and its model compounds have focused primarily on topics such as UVRR excitation profiles,24–26 excited-state relaxation rates and saturation spectra,27,28 and photoinduced transient radical species.29 Here, we present a systematic analysis of UVRR spectra of tryptophan derivatives to illustrate spectral markers for hydrogen bonding, environment polarity, and cation-π interactions of this unique amphiphilic residue under resonance conditions; these results are compared with previous off-resonance Raman data. In addition to this comparison, we also report new spectral markers that can be utilized to interpret UVRR spectra. The significance of the current work is the expansion of UVRR to membrane-associated proteins. Because of the enrichment of aromatic amino acids in membrane proteins and the large changes in environment and structure that are expected upon protein insertion into a bilayer, UVRR is an ideal tool for studies of membrane protein folding. Data and analyses presented here help differentiate tryptophan residues that are solvent-exposed, bound to lipid headgroups, or buried in the hydrophobic core of a lipid membrane with or without a hydrogen bonding partner. Results for the widely studied membrane toxin melittin as well as a membrane-associated molecule, tryptophan octyl ester, are presented to illustrate the successful application of UVRR spectroscopy in studying membrane-associated biomolecules. Materials and Methods Chemicals. N-Acetyl tryptophan ethyl ester (NATEE) was purchased from TCI America, tryptophan octyl ester (TOE) was obtained from Chem Impex International, 1,3-dimethyl-3,4,5,6tetrahydro-2(1H)-pyrimidinone (DMPU) was purchased from Sigma Aldrich, and deuterated water (>99%) was obtained from Spectra Stable Isotopes. All other tryptophan derivatives, solvents, and potassium phosphate salts were purchased from Fisher Scientific. Melittin was purchased from Axxora (San Diego, CA), and neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC) and anionic lipid 1-palmitoyl-2oleoyl-sn-glycero-3-[phospho-rac(1-glycerol)] (sodium salt, POPG) were obtained from Avanti Polar Lipids as chloroform solutions. Compounds were used as received without further purification. If necessary, solvents were dried with molecular sieves. Tryptophan derivative concentrations were 10-50 mM for all Raman experiments. TOE and melittin UVRR samples contained potassium phosphate buffer at pH 7.3. TOE concentrations were 40 µM in phosphate buffer and 15 µM in POPC vesicles. Melittin concentrations were 40 µM in each sample. Vesicle Preparation. To make 2:1 molar ratio POPC/POPG vesicles, a chloroform solution containing 5 mg of POPG was added to a chloroform solution containing 10 mg of POPC. The resulting solution of anionic lipid and a solution of 10 mg neutral POPC lipid were dried under a stream of argon. Dried lipids were resuspended in 20 mM potassium phosphate buffer using a bath sonicator. The vesicles were formed by extruding the lipid suspension eleven times through a polycarbonate filter with
Schlamadinger et al. pore size 200 nm (100 nm for TOE experiments) using a liposome extruder. This vesicle solution was filtered through a 0.45 µm filter and passed through a gravity-driven desalting column (BioRad). The first 3 mL elution was discarded, and the second 3 mL elution containing vesicle was collected and allowed to equilibrate for 2 h at 37 °C. This aliquot was confirmed to contain a majority (>70%) of ∼200 nm vesicles via dynamic light-scattering measurements. The final lipid concentration used in the experiments was 1 mg/mL. Steady-State Fluorescence Spectroscopy. Tryptophan fluorescence spectra were obtained on a Jobin Yvon Horiba Fluorolog-3 spectrofluorometer. The excitation wavelength was 280 nm for tryptophan derivatives and 290 nm for melittin and TOE, and the entrance and exit bandpass was 3 nm. Fluorescence spectra were recorded at a constant temperature of 20 °C. Raman Spectroscopy. The off-resonance Raman spectra were acquired with 514 nm excitation from a mixed-gas Kr-Ar ion laser. The 50 mW beam was focused onto a 1.8 mm o.d., 1.4 mm i.d. capillary tube and collected using a 90° scattering geometry as described previously.30 Scattered light was focused onto a 100 µm entrance slit, and Rayleigh light was rejected using a 514 nm long-pass edge filter (Semrock RazorEdge). Raman scattered light was dispersed in an f/6.9, 0.75 m spectrograph equipped with a 1200 grooves/mm grating and imaged onto a Peltier-cooled CCD. The spectral bandpass was 10 cm-1, and accuracy as determined by ethanol calibration was (1 cm-1. The precision based on repeatability was (1 cm-1. UV Resonance Raman Spectroscopy. The UVRR setup has been described elsewhere.31 Briefly, vibrational spectra of melittin and tryptophan derivatives were obtained by setting the fundamental laser wavelength to 920 nm to generate a 230 nm excitation beam. For tryptophan derivatives, a typical sample volume of 4 mL was flowed through a 200 µm i.d., 350 µm o.d., vertically mounted, fused-silica capillary at a rate of 0.40 mL/min to ensure fresh sample for each laser pulse. The capillary size for melittin and TOE experiments was 100 µm i.d., 160 µm o.d., and the flow rate was 0.16 mL/min. The UV power was ∼4 mW at the sample. Ten 1-min spectra were collected and summed for all samples with the exception of TOE, which required 30-60 min of acquisition time. UVRR spectra of all appropriate blank solutions were also collected and subtracted from the corresponding tryptophan derivative, TOE, and melittin spectra. Accuracy was determined using standard ethanol peaks and was found to be ( 2 cm-1. The bandpass for the Raman experiment was ∼8 cm-1. Overlapping peaks were decomposed into Gaussian bands using a leastsquares fitting routine. The precision based on repeatability was ( 2 cm-1. Correlation Analysis. The correlation coefficient, r, is defined as32
r)
∑ (xi - jx)(yi - jy) √ ∑ (xi - jx)2(yi - jy)2
where the xi are the values of the solvent parameter of interest (e.g., π*), jx is the average of the xi, the yi are the values of the spectroscopic parameter of interest (e.g., RFD), and jy is the average of the yi. The correlation coefficient, r, is a value between -1 and 1; values close to -1 indicate negative correlation, and values close to +1 indicate positive correlation. Values near 0 indicate no correlation. Probability is defined as
UV Resonance Raman Spectrum of Tryptophan
Figure 1. UVRR (230 nm excitation) and off resonance Raman spectra (514 nm excitation) of NATEE (A), NATA (B), skatole (C), and indole (D) in methanol. Tryptophan derivative UVRR spectra were normalized to the W18 band intensity; off-resonance data were arbitrarily scaled. All spectra are offset for clarity. Strong methanol peaks obscured signal in the regions of the off-resonance data marked by an asterisk.
the likelihood that the same number of measurements of two uncorrelated variables of x and y would produce a correlation coefficient with |runcorrelated| g |r|.32 Results Raman Spectra of Model Compounds with 230 and 514 nm Excitation Wavelengths. Raman spectra of 10-50 mM L-tryptophan (L-Trp), N-acetyl L-tryptophan ethyl ester (NATEE), N-acetyl L-tryptophanamide (NATA), 3-methylindole (skatole), and indole were acquired with incident wavelengths 230 and 514 nm. These derivatives, illustrated in the Supporting Information, were chosen because of the abundance of prior experimental data (L-Trp and NATA), solubility in organic solvents (NATEE, skatole, and indole), and extensive vibrational mode analysis (skatole and indole). NATEE, L-Trp, and NATA serve as models of the biologically relevant amino acid, whereas skatole and indole exhibit the greatest solubility in membranelike organic solvents. Concentration-dependence experiments revealed no changes in peak positions or relative intensities over the relevant range of concentrations in methanol. Raman spectra of each derivative in methanol are shown in Figure 1; a spectrum of L-Trp is shown in the Supporting Information and is consistent with prior reports. NATEE and NATA exhibit nearly identical spectra in terms of peak positions and relative intensities; skatole also displays similar bands, but indole spectra deviate substantially from these other tryptophan derivative spectra. Comparison of the on- and off-resonance Raman spectra for a given tryptophan derivative reveals that peak positions are similar, with less than 4 cm-1 difference for corresponding peaks. However, as expected, there are variations in relative band intensities between on- and off-resonance Raman spectra.33 The Fermi doublet intensity ratios also vary with excitation wavelength and tryptophan derivative. The Fermi doublet ratio is defined as RFD ) I∼1360 cm-1/I∼1340 cm-1 and was determined by directly obtaining intensity values from the spectra as well as
J. Phys. Chem. B, Vol. 113, No. 44, 2009 14771 by Gaussian decomposition of the overlapping bands. The RFD values from direct intensities are summarized in Table 1 for NATEE and indole on- and off-resonance and in different solvents; skatole RFD values are not resolved and therefore not presented here. Solvent properties34,35 of polarity/polarizability (π*), normalized polarity (ETN), and dielectric constant (εr) as well as the wavelengths of maximum fluorescence emission for indole are also listed. Fermi doublet values in off-resonance spectra of NATEE and indole are similar to those reported previously.21 Graphs of UVRR RFD as a function of the solvent properties π* and ETN along with representative data are shown in Figure 2. Linear fits to the data are also included. Decomposition of the Fermi doublet region resulted in slightly different RFD values; with the exception of dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO), the RFD values obtained via decomposition of the UVRR spectra for NATEE in various solvents were within 10% of the values obtained when direct intensities were utilized. The peak positions in the NATEE UVRR Fermi doublet shifted in some solvents; the low-energy peak varied from 1340 to 1346 cm-1, and the high-energy peak varied from 1363 to 1365 cm-1. Correlation coefficients between spectroscopic observables (UVRR RFD and emission λmax) and solvent parameters (π*, ENT , and hydrogen bond acceptor value β) are summarized in Table 2. The correlations between UVRR RFD and the solvent parameters π* and ETN are significant, with three of four of the probability values less than 2.1%. The correlation between RFD and ETN in the case of NATEE is not as strong (probability 12.9%). In contrast, analysis of RFD and β show that these parameters are unlikely to be correlated, with probabilities of 28.0% or 45.7%. In addition, the fact that the r values are of the opposite sign for NATEE and indole supports a lack of correlation between RFD and β. Analogous analysis of the fluorescence maximum λmax and solvent parameters indicate that λmax is strongly correlated to all three parameters: π*, ETN, and β. Graphs of RFD vs β as well as of λmax values are included as Supporting Information. UVRR Spectra of Tryptophan and Derivatives in H2O and D2O. UVRR spectra of NATEE, skatole, and indole in H2O and D2O are shown in Figure 3. These spectra are similar to previously published spectra15,36–38 and illustrate peaks that involve large displacement of the N-H moiety on the indole ring. Significant changes are observed for the W17, W8, and W4 peaks, with downshifts of 19, 19, and 10 cm-1, respectively, for skatole; the W17 and W4 peaks have been shown to be sensitive markers for hydrogen-bonding strength.22,23 Other significant spectral changes are observed in the W12 and W10 regions upon deuteration. Bands that are minimally perturbed upon deuteration include the intense W3 and W16 peaks. In D2O, the UVRR Fermi doublet shifts to lower frequency and is less resolved than in H2O. This behavior of the Fermi doublet in the UVRR spectra is consistent with previous on- and offresonance spectra.15,21 UVRR Spectra of Skatole with Solvent Titrations. The effects of hydrogen bonding of the indole N-H moiety on the resonance Raman spectrum was probed with skatole. Because skatole does not possess the backbone amide group, these solvent titrations reflect localized changes of the indole ring. Figure 4 shows UVRR spectra of skatole in mixtures of cyclohexane and dioxane to illustrate spectral changes associated with hydrogen bonding in a nonpolar environment; both solvents are nonpolar and aprotic but exhibit different hydrogen bond acceptor basicities based on the Kamlet-Taft β scale (cyclohexane β ) 0, dioxane β ) 0.37).39 This variation in hydrogen
14772
J. Phys. Chem. B, Vol. 113, No. 44, 2009
Schlamadinger et al.
TABLE 1: Solvent Polarity/Polarization Scales π* and ETN, Dielectric Constants εr,35 and Spectroscopic Parameters RFD and Emission Maxima (λfluo) of NATEE and Indolea solvent
π*
ETN
εr
UV RFDb
UV RFDc
Vis RFDb
water DMPU DMSO formamide DMF cyclohexanone acetonitrile methanol benzene ethanol toluene dioxane cyclohexane hexanes
1.09 1.08 1.00 0.97 0.88 0.68 0.66 0.60 0.55 0.54 0.49 0.49 0.00 -0.11
1.00 0.35 0.44 0.78 0.39 0.28 0.46 0.76 0.11 0.65 0.10 0.16 0.01 0.01
78 36 46 110 37 16 36 33 2.3 25 2.4 2.2 2.0 1.9
1.11 1.42 1.28 1.33 1.49 1.68 1.47 1.56 1.41 1.52 n.a. 1.50 n.a. n.a.
0.29 0.67 0.46 0.39 0.42 0.62 0.36 0.46 0.78 0.62 0.78 0.46 0.81 0.75
0.97 n.a. 1.21 n.a. n.a. n.a. 1.03 1.33 1.21 n.a. n.a. 0.98 n.a. n.a.
λfluo (nm)c 352 n.a. 334 340 329 n.a. 326 335 311 335 323 322 300 300
a RFD is defined as the ratio of intensities of the Fermi doublet at ∼1360 to ∼1340 cm-1 (I∼1360/I∼1340). b Values are for NATEE. c Values are for indole. DMPU, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone; DMSO, dimethyl sulfoxide; DMF, dimethyl formamide; UV, ultraviolet (230 nm excitation); Vis, visible (514 nm excitation); n.a., not acquired or not available due to solvent interference.
Figure 2. UVRR Fermi doublet RFD values and linear regression lines for indole (b) and NATEE (O) in nonhalogenated solvents as a function of solvent Kamlet-Taft π* and polarity ENT scale. Representative spectra and Gaussian decomposition are shown as insets.
TABLE 2: Correlation Coefficients (r) That Indicate Strength of Correlation between Spectral Markers and Solvent Parametersa spectral marker indole RFD NATEE RFD indole λmax
solvent parameter
correlation coefficient (r)
probability (%)
π* ETN β π* ETN β π* ETN β
-0.65 -0.77 -0.31 -0.68 -0.49 0.25 0.89 0.92 0.61
1.1
