Vacuum-Ultraviolet Circular Dichroism Spectra of Escherichia coli

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Vacuum-Ultraviolet Circular Dichroism Spectra of Escherichia coli Dihydrofolate Reductase and Its Mutants: Contributions of Phenylalanine and Tyrosine Side Chains and Exciton Coupling of Two Tryptophan Side Chains Eiji Ohmae, Suguru Tanaka, Yurina Miyashita, Katsuo Katayanagi, and Koichi Matsuo J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07480 • Publication Date (Web): 25 Sep 2015 Downloaded from http://pubs.acs.org on September 26, 2015

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The Journal of Physical Chemistry

Vacuum-Ultraviolet Circular Dichroism Spectra of Escherichia coli Dihydrofolate Reductase and Its Mutants: Contributions of Phenylalanine and Tyrosine Side Chains and Exciton Coupling of Two Tryptophan Side Chains

Eiji Ohmae,†,*, Suguru Tanaka,† Yurina Miyashita,† Katsuo Katayanagi,† and Koichi Matsuo‡

†

Department of Mathematical and Life Sciences, Graduate School of Science,

Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8526, Japan ‡

Hiroshima Synchrotron Radiation Center, Hiroshima University,

Higashi-Hiroshima, Hiroshima 739-0046, Japan

*Corresponding author

Tel/Fax: +81-82-424-7389; E-mail: [email protected]

Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; CD, circular dichroism; DHF, dihydrofolate; DHFR, dihydrofolate reductase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TDE, 20 mM Tris-hydrochloride (pH 8.0) containing 0.1 mM EDTA and 0.1 mM dithiothreitol (buffer); UV, ultraviolet; VUV, vacuum ultraviolet.

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ABSTRACT: Vacuum-ultraviolet (VUV) circular dichroism (CD) spectroscopy has recently been used for secondary structure analysis of proteins. However, the contribution of aromatic side chains to protein VUV CD spectra is unresolved. In this report, VUV CD spectra of 10 Escherichia coli dihydrofolate reductase (DHFR) mutants, in which each phenylalanine or tyrosine residue was mutated to leucine, were measured down to 175 nm at 25 °C and pH 8.0 to elucidate the contributions of these aromatic side chains to the high-energy transitions of peptide bonds. The VUV CD spectra of these mutants were different from the spectrum of the wild-type protein, indicating that the contribution of the phenylalanine and tyrosine side chains of DHFR extends to the VUV region. Further, the VUV CD spectrum and the folate- or NADP+-induced spectral change of F103L mutant DHFR indicated a modification and regeneration of exciton coupling between the Trp47 and Trp74 side chains, respectively, suggesting exciton coupling may also contribute to the CD spectrum of DHFR in the VUV region. These results should be useful for theoretically characterizing the contribution of aromatic side chains to protein CD spectra, leading to the improvement of protein secondary-structure analysis by VUV CD spectroscopy.

INTRODUCTION Circular dichroism (CD) in the far-ultraviolet (UV) region is very sensitive to the backbone structure of a protein, and this type of spectroscopy has been widely used for secondary-structure analysis of proteins. However, the far-UV CD spectra of some proteins such as bacteriophage fd 1, barnase 2, bovine pancreatic trypsin inhibitor (BPTI) 3, and Escherichia coli dihydrofolate reductase 2 ACS Paragon Plus Environment

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(DHFR) 4 are strongly affected by the contributions of aromatic side chains (tryptophan, phenylalanine, and tyrosine), because these aromatic chromophores have CD intensity from fully allowed π–π* transitions and exciton coupling of these transitions in this wavelength region (approximately 180–230 nm) 5, 6. Exciton coupling exhibits one intense absorption band in tertiary structure of a protein when two identical aromatic chromophores of the protein are in close proximity. In such case, two excited states of the pair, whose energies are equally increased or decreased from the excited state of each chromophore, are generated resulting from exciton mixing of the transitions. Since the energetic separation of the excited-state pair is generally smaller than the width of the individual chromophore transitions, the resulting two bands are overlap with some cancellation, and characteristic two CD peaks having opposite signs and equal intensities are observed 6. Protein CD spectra were recently calculated from the three-dimensional coordinates of backbone structure and aromatic side chains using CD theory 7. This method was applied to CD calculations of BPTI and DHFR with mutations of their aromatic residues, and it suitably characterized the contributions of the aromatic side chains to their far-UV CD spectra 7, indicating the usefulness of comparative studies between experimental and theoretical CD spectra. In addition to contributing to the CD spectrum in the far-UV region, aromatic side chains also contribute to the CD spectrum in the vacuum-ultraviolet (VUV) region, although VUV CD spectroscopy using synchrotron radiation can analyze the secondary structure of proteins more accurately than conventional far-UV CD spectroscopy 8–10. Bulheller et al. showed that the differences between 3 ACS Paragon Plus Environment


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theoretical and experimental VUV CD spectra of proteins are reduced when the contributions of aromatic side chain transitions are incorporated into the theory 11. The first experimental evidence for the contributions of aromatic side chains to protein VUV CD spectra was found in the spectral change of DHFR mutants in which tryptophan residues were replaced with leucine, phenylalanine, or valine 12

. In addition, Matsuo et al. recently showed that phenylalanine−tyrosine

interactions in the amyloid fibrils of peptides contribute to experimental VUV CD spectra using CD theory 13. However, there is only a limited amount of explicit experimental data on the contributions of aromatic side chains to VUV CD spectra. DHFR is a model protein whose structure 14, 15, stability 16, 17, dynamics 18–20, and function 21, 22 have been well characterized. DHFR is 159 residues in length and contains five tryptophan, six phenylalanine, and four tyrosine side chains (Fig. 1). Previously, we found that the VUV CD spectrum of DHFR was significantly affected by mutation of the tryptophan residues 12. Moreover, exciton coupling between the Trp47 and Trp74 side chains, which are located in the adenosine binding subdomain (residues 38–106), was found to contribute to the far-UV region (i.e., 210–240 nm) of the CD spectrum of DHFR 23. Conversely, most phenylalanine and tyrosine residues, except Tyr100 and Phe103, are located in another subdomain, the discontinuous loop subdomain (residues 1–37 and 107– 159). Currently, there are no data for quantitatively evaluating the contributions of phenylalanine and tyrosine side chains to the VUV CD spectra of proteins. In this study, we measured VUV CD spectra of single mutant DHFRs by replacing each phenylalanine or tyrosine residue with leucine down to 175 nm to elucidate 4 ACS Paragon Plus Environment

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the contributions of these side chains to the high energy transitions of peptide bonds. The results of this study clearly indicated that the phenylalanine and tyrosine side chains, in addition to exciton coupling between two tryptophan side chains, contributed to the CD spectrum of DHFR in the VUV region. (Figure 1)

EXPERIMENTAL METHODS Construction and Purification of Mutant DHFRs. Plasmid construction and protein purification methods for the mutant DHFRs have been described previously 24. The DNA sequences of the synthetic oligonucleotide primers used in this study are shown in Table 1. The entire DNA sequences of all mutant genes were evaluated to avoid unexpected mutations. The purified proteins were fully dialyzed against 20 mM Tris-hydrochloride (pH 8.0) containing 0.1 mM EDTA and 0.1 mM dithiothreitol (TDE buffer) before spectroscopic measurements. The purity of the mutant DHFRs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the purified DHFR mutants was determined spectrophotometrically with a molar extinction coefficient of 31,100 M-1 cm-1 at 280 nm 21 for the wild-type and phenylalanine-mutant DHFRs, because the absorption of a phenylalanine side chain at this wavelength is negligible. The molar extinction coefficient was corrected to 29,610 M-1 cm-1 for the tyrosine-mutant proteins.



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Table 1. Oligonucleotide primers used in PCR amplification of mutant DHFR genes Primer

Sequence

p35-F

5′-GGGGATCCGCTCTTGACAATTAGTTAACTATTTGT-3′

p35-R

5′-GAGGATCCTTAACGACGCTCGAGGATTTCGAAACA-3′

pF31L

5′-GGTGTTGCGTTTAAGCCAGGCGAGATC-3′

pF103L

5′-GTTTATGAACAGCTCTTGCCAAAAGCG-3′

pF125L

5′-GGCGACACCCATCTTCCGGATTACGAG-3′

pF137L

5′-TGGGAATCGGTACTCAGCGAATTCCAC-3′

pF140L

5′-GTATTCAGCGAACTCCACGATGCTGAT-3′

pF153L

5′-CATAGCTATTGTCTCGAAATCCTCGAG-3′

pY100L

5′-CGGCGGACGCGTTCTTGAACAGTTCTTGC-3′

pY111L

5′-GCGCAAAAGCTTCTTCTGACGCATATC-3′

pY128L

5′-CATTTTCCGGATCTCGAGCCGGATGAC-3′

pY151L

5′-AACTCGCATAGCCTTTGTTTCGAAATC-3′

Enzyme Assay. The enzymatic activity of mutant DHFRs was measured using a V-560 spectrophotometer (JASCO, Inc., Tokyo, Japan) as described previously 24

. The temperature was maintained at 25 °C with a circulating thermobath

(NESLAB RTE-5; Thermo Fisher Scientific, Waltham, MA, USA). The solvent used was TDE buffer. The concentrations of both dihydrofolate (DHF; Sigma-Aldrich, St. Louis, MO) and NADPH (Oriental Yeast, Tokyo, Japan) were kept at 50 µM, and were determined spectrophotometrically using molar extinction coefficients of 28,400 M-1 cm-1 at 282 nm and 6,200 M-1 cm-1 at 339 6 ACS Paragon Plus Environment

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nm, respectively 25. The enzyme concentration was 10 nM. The enzymatic reaction was initiated by adding the DHF solution to the enzyme-NADPH mixture, and the initial velocity of the reaction was determined using a differential molar extinction coefficient of 11,800 M-1 cm-1 at 340 nm 21. VUV CD Spectra. VUV CD spectra of the wild-type and mutant DHFRs were measured from 260 to 175 nm using a VUV CD spectrophotometer constructed at the Hiroshima Synchrotron Radiation Center (HiSOR, Higashi-Hiroshima, Japan) and an assembled-type optical cell at 25 °C 26, 27 under a nitrogen atmosphere. The optical path length of the cell was adjusted to 10 µm using a Teflon spacer. The solvent used was TDE buffer, and the protein concentration was 400 µM. When the folate or NADP+ (Oriental Yeast) concentration dependencies of the VUV CD spectrum of the F103L mutant were measured, the protein concentration was set to 770 µM. The concentrations of folate and NADP+ were set to 0, 400, and 880 µM, which were determined spectrophotometrically using molar extinction coefficients of 27,000 M-1 cm-1 at 282 nm and 18,000 M-1 cm-1 at 260 nm, respectively 25. The VUV CD spectrum of the same concentration of ligand was subtracted from that containing the protein and ligand. Far-UV CD Spectra and Equilibrium Dissociation Constants. To determine the equilibrium dissociation constants, the folate and NADP+ concentration dependency of the far-UV CD spectrum of F103L mutant DHFR was measured at 25 °C using a JASCO J-720W spectropolarimeter (JASCO, Inc.) as described previously 28. The buffer used was the TDE buffer and the protein concentration was 10 µM. The protein and ligands were mixed and equilibrated for 1 h before 7 ACS Paragon Plus Environment


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measurements. The observed molar ellipticity at 230 nm, [θ]230, was plotted against the ligand concentration and fitted to the following equation using nonlinear least-squares analysis 28: [θ]230 = [θ]0E + ∆[θ] [(E + L + Kd) – {(E +L + Kd)2 – 4EL}0.5] / 2

(1)

Here, [θ]0 is the molar ellipticity of the protein without ligand, ∆[θ] is the molar ellipticity change between the protein–ligand complex and free protein, and E and L are the concentrations of the protein and ligand, respectively.

RESULTS AND DISCUSSION Purity and enzyme activity of mutants of DHFR. As shown in Fig. 2A, all mutant DHFR proteins were purified to show a prominent band on an SDS-PAGE gel. The relative enzymatic activities of the mutants were determined to be >50% of the wild-type protein, except for the F31L (32%) and Y100L (8%) mutants (Fig. 2B). Since enzymatic activity was measured at saturating substrate and cofactor concentrations, the reduced activity of these mutants relative to the wild-type protein was attributed to decreases in the kcat values. Since all the mutants could bind the substrate and cofactor, and exhibited enzymatic activity, they were considered to have the same fold and secondary structures as wild-type DHFR. (Figure 2) VUV CD Spectra. Fig. 3A shows the VUV CD spectra of wild-type DHFR at 25 °C and pH 8.0 in comparison with the previous result using 10 mM potassium phosphate buffer at the same temperature and pH 7.0 12. Both spectra at pH 7.0 and 8.0 are similar; however, the shoulder associated with the positive peak 8 ACS Paragon Plus Environment

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shown in the region of 180–185 nm was less obvious for the spectrum recorded at pH 8.0 than the data at pH 7.0. This difference might have originated from the difference in pH or binding of a potassium cation, since the binding of an inorganic cation to wild-type DHFR has been reported previously 24. (Figure 3) Fig. 3B shows the VUV CD spectra of wild-type DHFR and the six phenylalanine mutants at 25 °C and pH 8.0. The VUV CD spectra of the F137L, F140L, and F153L mutants were similar to that of the wild-type spectrum; however, their positive peak intensities were slightly different: 9,790 deg cm2 dmol-1 at 193 nm (F137L), 10,610 deg cm2 dmol-1 at 194 nm (F140L), and 9,090 deg cm2 dmol-1 at 193 nm (F153L). Conversely, the positive peak in the spectra of the F31L and F125L mutants was clearly broadened with blue-shifted peak wavelengths and weakened intensities: 6,830 deg cm2 dmol-1 at 190 nm (F31L), and 7,350 deg cm2 dmol-1 at 191 nm (F125L). Similarly, the shape of the negative peak of these two mutants was also broadened with peak wavelengths and intensities of -6,620 deg cm2 dmol-1 at 217 nm (F31L) and -7,190 deg cm2 dmol-1 at 222 nm (F125L). Compared with the small spectral change of wild-type DHFR at pH 7.0 and 8.0 (Fig. 3A), these results clearly showed that the phenylalanine side chains contribute to the CD spectrum of DHFR in the far-UV and VUV regions. In particular, the VUV CD spectrum of the F103L mutant was quite different from that of the wild-type protein. Both the positive and negative peaks were shifted 8 nm to 186 and 228 nm with peak intensities of 11,020 and -9,830 deg cm2 dmol-1, respectively. This characteristic spectrum might be ascribed to modifications of exciton coupling between the Trp47 and Trp74 side chains because similar 9 ACS Paragon Plus Environment


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far-UV CD spectra were observed previously for Met42 DHFR mutants in which exciton coupling was modified 28. This is possible because Phe103 and these tryptophan residues are parts of the same adenosine binding subdomain, and side chains of Phe103 and Trp47 contact with those of Ile41 and Met42, respectively, constructing a hydrophobic core in the tertiary structure of DHFR. The VUV CD spectra of wild-type DHFR and the tyrosine mutants are shown in Fig. 3C. Although the Y128L mutant showed a similar spectrum to wild-type DHFR with a reduction in the intensity of the positive peak, the other three mutants, Y100L, Y111L, and Y151L, showed broadened positive peaks accompanying clearly blue-shifted peak wavelengths and significant decreases in peak intensities of 5,930 deg cm2 dmol-1 at 190 nm (Y100L), 9,310 deg cm2 dmol-1 at 191 nm (Y111L), and 10,450 deg cm2 dmol-1 at 191 nm (Y151L). Additionally, the negative peak of the VUV CD spectrum of the Y100L mutant was also broadened. Although enlarged negative peak around 205 nm of the Y100L mutant suggested the increase of unfolded fraction under the experimental condition in its equilibrium between native and unfolded states, fluorescence spectrum of this mutant showed only small changes in the peak wavelength and intensity compared to those of the wild-type protein, indicating that the mutant has similar tertiary structure and population of the native conformers with the wild-type protein (data not shown). Besides, Liu et al. reported that the hydroxyl group of Tyr100 side chain played an important electrostatic role on the protonation of DHF in the catalytic reaction of DHFR 22. Therefore, the reduced enzymatic activity of this mutant was not derived from perturbation of the overall structure. These results clearly indicated that the 10 ACS Paragon Plus Environment

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tyrosine side chains also contributed to the CD spectrum of DHFR in the far-UV and VUV regions. VUV CD Difference Spectra between Wild-Type and Mutant DHFRs. Fig. 4 shows the VUV CD difference spectra at 25 °C and pH 8.0 calculated by subtraction of the spectra of the phenylalanine (A) or tyrosine (B) mutants from the wild-type DHFR spectrum. As shown in this figure, the difference spectra for the phenylalanine and tyrosine mutants typically showed positive and negative molar ellipticities for the VUV and far-UV regions, respectively, although several exceptional examples and variation of the spectral shapes indicated that the contribution of the aromatic side chains was highly position dependent. (Figure 4) In a previous study, we reported that VUV CD difference spectra between phenylalanine and leucine mutants at sites 22 (W22F and W22L) and 74 (W74F and W74L) showed two positive peaks at 197 and 220 nm 12, which is similar to the CD spectrum of N-acetyl-L-phenylalanine amide 29. The peak at 197 nm was attributed to degenerate Ba and Bb bands of the phenyl ring, and the 220 nm peak was assigned to an La band 6. In the present study, two positive peaks were observed only in the difference spectrum of the F103L mutant, which had two strong intensity peaks of 5,300 and 3,800 deg cm2 dmol-1 at 195 and 230 nm, respectively (Fig. 4A). However, both peaks might be attributable to modifications of exciton coupling between the Trp47 and Trp74 side chains, as discussed in the next section. Conversely, the difference spectra for the F31L and F125L mutants were essentially identical, with one large positive peak at 195 nm with intensities of 11 ACS Paragon Plus Environment


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6,300 and 5,000 deg cm2 dmol-1, respectively, and an accompanying shoulder at 180–190 nm. Additionally, a single relatively small negative peak of -1,600 deg cm2 dmol-1 at 220 nm and -1,000 deg cm2 dmol-1 at 217 nm was observed for the F31L and F125L mutants, respectively. Therefore, the contributions of the B and La bands of the phenyl rings were clearly observed in the difference spectra for these mutants in the VUV and far-UV regions, respectively, although the La band contributed negatively. The VUV CD difference spectra for the other three phenylalanine mutants, F137L, F140L, and F153L, were small, with molar ellipticity differences between the spectra of the wild-type protein and the three mutants within ±2,200 deg cm2 dmol-1 at all wavelengths, suggesting relatively small contributions from the phenyl rings. For the tyrosine mutants shown in Fig. 4B, the VUV CD difference spectrum for the Y100L mutant also showed one large positive peak of 6,600 deg cm2 dmol-1 at 196 nm with a shoulder at 180–190 nm, and one relatively small negative peak of -1,300 deg cm2 dmol-1 at 231 nm. Similar differences were observed for the other three mutants, although the positive peak intensities were significantly weaker with values of 3,000 deg cm2 dmol-1 at 195 nm (Y111L), 2,800 deg cm2 dmol-1 at 189 nm (Y128L), and 2,500 deg cm2 dmol-1 at 201 nm (Y151L). Large positive peaks in VUV region of the difference spectra for the Y100L, F31L, and F125L mutants suggested additional contributions such as the exciton coupling generated by the aromatic−aromatic and/or the backbone−aromatic interactions 3, 13

to the CD spectrum of DHFR. Since the distance between the centroid of the

phenol ring of Tyr100 and that of the phenyl ring of Phe125 in the crystal 12 ACS Paragon Plus Environment

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structure of DHFR is only 5.1 Å, coupling of these aromatic rings would contribute to the VUV CD spectrum. Conversely, there is no such aromatic ring near Phe31, thus the contribution of backbone−aromatic coupling is expected. Compared with the phenyl ring, the phenol ring of tyrosine residues requires lower energy for transitions to the La, Ba, and Bb states 6. As a result, N-acetyl-L-tyrosine amide gives rise to a CD spectrum that is similar to the spectrum of N-acetyl-L-phenylalanine amide with an accompanying slight red-shift of the two positive peaks at approximately 202 and 230 nm 29. Thus, the positive and negative peaks shown in the difference spectra for these tyrosine mutants can be attributed to the contributions of the B and La bands of the phenol ring, respectively, as in the case of the phenylalanine mutants. Compared with the phenylalanine mutants, the negative peak wavelengths of the difference spectra for the Y100L, Y111L, and Y151L mutants (231 nm) were clear, while that for the Y128L (221 nm) mutant was slightly red-shifted from those for the F31L (220 nm) and F125L (217 nm) mutants. However, the positive peak wavelengths of the difference spectra for Y100L (196 nm) and Y111L (195 nm) were almost the same as that for Y128L (189 nm), which was 6-nm blue-shifted from those for the F31L and F125L mutants (195 nm), although that for Y151L (201 nm) was 6-nm red-shifted. Therefore, the presence of some other contributions can be presumed in the difference spectra of these tyrosine DHFR mutants. CD Spectral Change of F103L DHFR Induced by Ligand Binding. As described above, our results suggest that the CD spectral change observed around 228 nm in the F103L mutant (Fig. 3B) might be attributable to the 13 ACS Paragon Plus Environment


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modification of exciton coupling between the Trp47 and Trp74 side chains 23. We previously found that the modified exciton coupling of the M42I mutant was regenerated by the binding of a substrate, folate 28, and the F103L mutant had almost comparable (approximately 80%) enzymatic activity with the wild-type enzyme (Fog. 2B), so the substrate or cofactor can be expected to induce the spectral change of the F103L mutant relative to the wild-type protein. Therefore, we measured the folate concentration-dependence of the far-UV CD spectrum of the F103L mutant as shown in Fig. 5A. The negative peak intensity of the spectrum at 228 nm had clearly decreased and the intensities around 218 nm were increased in the opposite direction by the addition of folate with isoelliptic points at 209, 224, and 240 nm. Such a spectral change depending on the concentration of folate was the same as those observed in the M42I mutant 28. These results clearly indicate that exciton coupling of Trp47 and Trp74 side chains was modified by the F103L mutation and the addition of folate enabled these tryptophan side chains to adopt an orientation that is similar to that of the wild-type protein, which is suitable to exhibit the enzymatic activity. (Figure 5) A similar result was also observed for the NADP+ concentration-dependence of the far-UV CD spectrum of the F103L mutant; however, the isoelliptic points were slightly different with values of 206, 223, and 245 nm (Fig. 5B). This result indicates that the addition of NADP+ also regenerated exciton coupling between the Trp47 and Trp74 side chains, but the orientation of both side chains is different between the F103L-folate and F103L-NADP+ complexes.


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From the molar ellipticity change at 230 nm, we calculated dissociation constants, Kd, for both ligands using nonlinear least-squares fitting with Eq. 1, which was derived from a one-to-one binding model. The theoretical curves fitted well to the experimental results, indicating that the spectral change was induced by the binding of each ligand to the F103L mutant (Fig. 5C). The obtained Kd values were 23 ± 3 and 30 ± 3 µM for folate and NADP+, respectively. These values are slightly larger than the corresponding values for wild-type DHFR at pH 7.0 (1.5 ± 0.2 and 15.0 ± 2.3 µM, respectively) 30. A matter of concern is whether the ligand concentration-dependent spectral change is observed in the VUV region of the CD spectrum of the F103L mutant. To clarify this point, we measured the VUV CD spectra of the F103L-folate and F103L-NADP+ binary complexes. As shown in Fig. 6A, the VUV region of the CD spectrum of the F103L mutant clearly changed after the addition of folate. The positive peak at 186 nm decreased in intensity and the ellipticities around 195 nm, which match the positive peak of wild-type DHFR (Fig. 3A), increased as the concentration of folate increased with an isoelliptic point at 192 nm. Similar results were also observed for the F103L-NADP+ complex, with the isoelliptic point shifted to approximately 190 nm (Fig. 6B). (Figure 6) The inset of Fig. 6A shows the VUV CD difference spectra of the F103L mutant with or without folate. The molar ellipticity changes for the positive and negative peaks at 199 and 184 nm for the difference spectrum were similar, 3,150 and -2,820 deg cm2 dmol-1, respectively, with 880 µM folate, and 1,450 and -1,950 deg cm2 dmol-1, respectively, with 400 µM folate. From the Kd value between 15 ACS Paragon Plus Environment


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F103L and folate (23 ± 3 µM), the population of the F103L-folate binary complexes in the VUV CD samples were calculated as 50% and 89% for the 400 and 880 µM folate samples, respectively, which are proportional to the observed ellipticity changes. Therefore, these spectral changes in the VUV region appear to reflect the same phenomenon as in the far-UV region, that is, the contribution of exciton coupling between the Trp47 and Trp74 side chains. Similar results were also observed for the VUV CD difference spectra with or without NADP+, as shown in the inset of Fig. 6B. Since N-acetyl-L-tryptophan amide has a large negative CD peak at 195 nm in addition to a positive peak at 230 nm 29, exciton coupling could affect the VUV region of the CD spectrum of DHFR. This is the first observation of the contribution of exciton coupling of tryptophan side chains to the VUV region of a CD spectrum of a native protein. Thus, this result should be useful for theoretically characterizing the contributions of aromatic side chains to protein CD spectra, leading to the improvement of protein secondary-structure analysis by VUV CD spectroscopy.

CONCLUSIONS The phenylalanine and tyrosine side chains of DHFR contributed to the VUV region of the CD spectrum as well as to the far-UV region. These contributions were highly position dependent, but the characteristic positive and negative CD peaks around 230 and 184 nm, respectively, in the difference spectra of F103L mutant of DHFR were clearly attributed to the modification of exciton coupling between the Trp47 and Trp74 side chains. The results of this study should be useful for theoretically characterizing the contributions of aromatic side chains to 16 ACS Paragon Plus Environment

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protein CD spectra and the improvement of protein secondary-structure analysis by VUV CD spectroscopy, since the aromatic contributions to the CD spectra depress the accuracy of the analysis.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. Tel/Fax: +81-82-424-7389 Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported financially by a JSPS KAKENHI Grant (No. 24570186 to E.O.). The VUVCD experiments were performed with the approval of the Hiroshima Synchrotron Radiation Center (Proposal Nos. 13-B-49 and 14-B-4).



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Figure Captions Figure 1. Backbone cartoon drawing of the DHFR-NADP+-folate ternary complex (PDB code: 1rx2) 14. NADP+ (magenta) and folate (cyan) are shown as line models. The six phenylalanine, four tyrosine, and five tryptophan side chains are drawn as stick models with labels and are colored red, blue, and green, respectively. This figure was drawn using the program Pymol (http://www.pymol.org/).

Figure 2. (A) SDS-PAGE gel showing purified mutant DHFR proteins. Lanes 1 and 12: Molecular weight markers. Lanes 2 to 11: Purified DHFR mutant proteins of Y100L, Y111L, Y128L, Y151L, F31L, F103L, F125L, F137L, F140L, and F153L, respectively. (B) Relative enzymatic activity of the mutant DHFRs at 25 °C and pH 8.0. The activity of the wild-type protein was set to 100%.

Figure 3. (A) VUV CD spectra of wild-type DHFR at 25 °C and pH 8.0 (black) or 7.0 (red). The solvents used were 20 mM Tris-hydrochloride (pH 8.0) and 10 mM potassium phosphate (pH 7.0). Both solvents contained 0.1 mM EDTA and 0.1 mM dithiothreitol. The protein concentration was 400 µM. The spectrum at pH 7.0 was taken from Ohmae et al. 12. (B and C) VUV CD spectra of wild-type DHFR and the phenylalanine (B) or tyrosine (C) DHFR mutants at 25 °C and pH 8.0. (B) The line colors indicate wild-type (black), F31L (red), F103L (green), F125L (blue), F137L (cyan), F140L (magenta), and F153L (yellow). (C) The line colors indicate wild-type (black), Y100L (red), Y111L (green), Y128L (blue), and Y151L (cyan). 23 ACS Paragon Plus Environment


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Figure 4. VUV CD difference spectra between wild-type DHFR and the phenylalanine (A) or tyrosine (B) DHFR mutants at 25 °C and pH 8.0. The line colors of each panel are the same as in Fig. 3B and 3C, respectively.

Figure 5. Folate (A) and NADP+ (B) concentration-dependencies of the far-UV CD spectrum of F103L mutant DHFR at 25 °C and pH 8.0. The solvent used was TDE buffer. The protein concentration was 10 µM. Ligand concentrations were 0 (black), 1 (red), 3 (green), 5 (blue), 10 (cyan), 15 (magenta), 20 (yellow), 30 (purple), 50 (orange), and 100 (dark green) µM. (C) Ligand concentration-dependencies of molar ellipticity at 230 nm. The black and red circles indicate folate and NADP+, respectively. The lines indicate the least-squares fit to Eq. 1 (see EXPERIMENTAL METHODS).

Figure 6. Folate (A) and NADP+ (B) concentration-dependencies of the VUV CD spectrum of F103L mutant DHFR at 25 °C and pH 8.0. The solvent used was TDE buffer. The protein concentration was 770 µM. Ligand concentrations were 0 (black), 400 (red), and 880 (green) µM. Insets show VUV CD difference spectra with or without folate (A) or NADP+ (B).


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Tyr111

Trp74

Trp47

Phe103 Phe31 Trp30 Tyr100 Trp133 Phe125

Phe137 Phe153

Tyr128 Tyr151

Trp22

Phe140

Figure 1: E. Ohmae et al.



A kDa 1

2

3

4

5

6 7

8

9 10 11 12 kDa

150 75 50 37

150 75 50 37

25 20

25 20

15

15

10

10

B 100

Relative activity / %

80 60 40 20

1L F1 03 L F1 25 L F1 37 L F1 40 L F1 53 L Y 10 0L Y 11 1L Y 12 8L Y 15 1L

F3

ild

0 W

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DHFR mutants



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15 10

A

5 0 -1

-5

-3

2

[ ]×10 / deg cm dmol

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-10 15 10

B

5 0 -5 -10 15 10

C

5 0 -5 -10 180

200

220

240

Wavelength / nm

Figure 3: E. Ohmae et al. 26 ACS Paragon Plus Environment

260


6

A

-1

4

-3

2

[ ]×10 / deg cm dmol

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2 0 -2 -4

B

6 4 2 0 -2 180

200

220

240

Wavelength / nm


260

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5

A

-5

2


-1

0

-10 5

-3

B

0

-5

-10 250

240

230

220

210

200

2

[ ]230×10 / deg cm dmol

-1

Wavelength / nm -6

C -7 -8

-3

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


-9

-10 0

20

40

60

[Ligand] / M

80


100

260


15 -1

deg cm dmol

-3

3 2 1

2

[ ]×10 /

4

10

0 -1 -2

5

-3

-1

0

2

180

-5 -10 15

200

220

240

260

Wavelength / nm

A

5

2

deg cm dmol

-3

10

-1

4

[ ]×10 /

-3


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3 2 1 0

-1 -2 -3 180

200

220

240

260

Wavelength / nm

0 -5 -10

B

180

200

220

240

Wavelength / nm



260

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TOC image:

Phe103 Phe31 Tyr100 Phe125

Mutation to leucine

Phe137

0

F103L + Folate

-5

F103L

-10 15

Wild-type Y100L Y111L Y128L Y151L

10 5

F103L + NADP+

5

Tyr151

Phe153 Phe140

200 220 240 Wavelength / nm

260

180

200 220 240 260 Wavelength / nm

-5 -10 15 10 5 0

4 3 2 1 0 -1 -2 -3

-5

-5 -10

180

0

0

Tyr128

4 3 2 1 0 -1 -2 -3

D[θ ]×10-3 / deg cm2 dmol-1

5

10 [θ ]×10-3 / deg cm2 dmol-1

Wild-type F31L F103L F125L F137L F140L F153L

10

D[θ ]×10-3 / deg cm2 dmol-1

15

15

Tyr111 [θ ]×10-3 / deg cm2 dmol-1

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-10 180

200 220 240 Wavelength / nm

260


180 200 220 240 260 Wavelength / nm

Vacuum-Ultraviolet Circular Dichroism Spectra of Escherichia coli

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