Biochemistry 1987, 26, 1373-1 38 1 Washington, DC. Bhown, A. S., Mole, J. E., Wessinger, A., & Bennett, J. C. (1978) J . Chromatogr. 148, 532-535. Chang, J. Y. (1983) Methods Enzymol. 91, 455-466. Dayhoff, M. O., & Hunt, L. T. (1972) in Atlas of Protein Sequence and Structure (Dayhoff, M. O., Ed.) Vol. 5, pp 355-359, National Biomedical Research Foundation, Washington, DC. De Renaboles, M., Rogers, L., & Kolattukudy, P. E. (1 980) Arch. Biochem. Biophys. 205, 464-477. Gross, E., & Witkop, B. (1962) J . Biol. Chem. 237, 1856-1860. Hart, G. W., Brew, K., Grant, G. A., Bradshaw, R. A., & Lennarz, W. J. (1979) J . Biol. Chem. 254, 9747-9753. Hausinger, R. P., & Howard, J. B. (1982) J. Biol. Chem. 257, 2483-2490. Hayashi, R. (1977) Methods Enzymol. 47, 85-93. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., & Dryer, W. J. (1981) J . Biol. Chem. 256, 7990-7997. Hunkapiller, M. W. (1987) in Symposium of American Protein Chemists (Italien, J. J. L., Ed.) Plenum, New York (in press). Karplus, P. A., Walsh, K. A., & Herriot, J. R. (1984) Biochemistry 23, 6575-6583. Knudsen, J., Clark, S., & Dils, R. (1976) Biochem. J . 160, 683-69 1. Kulbe, K. D. (1974) Anal. Biochem. 59, 564-573. Libertini, L. J., & Smith, S. (1978) J . Biol. Chem. 253, 1393-1 401. Libertini, L. J., Linn, C. Y., & Smith, S. (1976) Fed. Proc., Fed. Am. SOC.Exp. Biol. 35, 1671. Mahoney, W. C . , & Hermodson, M. A. (1980) J. Biol. Chem. 255, 11199-11203.
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Mahoney, W. C., Smith, P. K., & Hermodson, M. A. (1981) Biochemistry 20, 443-448. Perham, R. N. (1978) in Techniques in Protein and Enzyme Biochemistry (Kornberg, H. L., Metcalfe, J. C., Northcote, D. H., Pogson, C. I., & Tipton, K. F., Eds.) Vol. B110, pp 1-39, Elsevier/North-Holland, Amsterdam. Pisano, J. J., & Bronzert, T. J. (1972) Anal. Biochem. 45, 43-59. Pohl, G., Kallstrom, M., Bergsdorft, N., Wallen, N., & Jornvall, H . (1984) Biochemistry 23, 3701-3707. Poulose, A. J., Rogers, L., Cheesbrough, T. M., & Kolattukudy, P. E. (1985) J . Biol. Chem. 260, 15953-15958. Randhawa, Z . I., Naggert, J., Blacher, R. W., & Smith, S. (1987) Eur. J . Biochem. 162, 577-581. Reimer, N. S., Yasunobu, K. T., Wei, Y. H., & King, T. E. (1983) Biochem. Biophys. Res. Commun. 110, 8-14. Safford, R., de Silva, J., Lucas, C., Windust, J. H. C., Shedden, J., James, C. M., Sidebottom, C. M., Slabas, A. R., Tombs, M. P., & Hughes, S. G. (1987) Biochemistry (preceding paper in this issue). Schroeder, W. A., Shelton, J. B., & Shelton, J. R. (1980) Hemoglobin 4, 551-558. Shoji, S . , Enicsson, L. H., Walsh, K. A., Fischer, E. M., & Titani, K. (1983) Biochemistry 22, 3702-3709. Smith, S., & Libertini, L. J. (1979) Arch. Biochem. Biophys. 196, 88-92. Smith, S., Stern, A., Randhawa, Z. I., & Knudsen, J. (1985) Eur. J . Biochem. 152, 547-555. Smith, S., Mikkelsen, J., Witkowski, A., & Libertini, L. J. (1986) Biochem. SOC.Trans. 14, 583-584. Witkowski, A., & Smith, S. (1985) Arch. Biochem. Biophys. 243, 420-426.
Structure and Dynamics of the Pfl Filamentous Bacteriophage Coat Protein in Micelles? R. A. Schiksnis, M. J. Bogusky, P. Tsang, and S . J. Opella* Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 191 04 Received September 12, 1986; Revised Manuscript Received November 6, 1986 ABSTRACT: The major coat protein of filamentous bacteriophage adopts its membrane-bound conformation
in detergent micelles. High-resolution 'H and I5NN M R experiments are used to characterize the structure and dynamics of residues 30-40 in the hydrophobic midsection of Pfl coat protein in sodium dodecyl sulfate micelles. Uniform and specific-site 15Nlabels enable the immobile backbone sites to be identified by their 'H/I5N heteronuclear nuclear Overhauser effect and allow the assignment of 'H and 15Nresonances. About one-third of the amide N-H protons in the protein undergo very slow exchange with solvent deuterons, which is indicative of sites in highly structured environments. The combination of results from 'H/15N heteronuclear correlation, 'H homonuclear correlation, and 'H homonuclear Overhauser effect experiments assigns the resonances to specific residues and demonstrates that residues 30-40 of the coat protein have a helical secondary structure. T e current level of understanding of biological processes involving membrane-bound proteins is not as detailed as for those involving cytoplasmic proteins. This is due, in large part, to the difficulty in applying methods capable of yielding +Thisresearch was supported by Grant AI-20770 from the NIH and Smith Kline & French Laboratories. The 400-MHz NMR spectrometer was obtained with Grant PCM-8207163 from the NSF. R.A.S. was supported by a Cell and Molecular Biology Training Grant.
0006-296018710426- 137 3$01,50/0
structural information at atomic resolution to membranebound proteins (Eisenberg, 1984). Although considerable progress has been made with diffraction (Leifer & Henderson, 1983) and N M R (Keniry et al., 1984) studies on describing the structure of bacteriorhodpsin in purple membrane, and with diffraction on the structure of porin of Escherichia coli outer membrane (Kleffel et al., 1985), only the recent diffraction results on the photoreaction center (Deisenhofer et al., 1985) crystallized from detergent (Michel, 1982) approach the 0 1987 American Chemical Society
1374
SCHIKSNIS ET A L .
BIOCHEMISTRY 10
1
G-V.1.D-T-S-A-V-O-S-A-I-T-D-G-D20 30 M-K - A- I - G -GY -I -V-G - A- L. V-I- L-A- V-A-G. 40
46
L-I-Y-S-M-L-R-K-A FIGURE 1: Amino acid sequence of Pfl coat protein (Nakashima et al., 1975). The 11 residues with very slow amide proton exchange rates are underlined.
resolution routinely obtainable for globular proteins (Richardson, 1981). N M R studies of membrane-bound proteins can be performed in two quite different ways, depending on whether solid-state N M R or solution N M R methods are employed. Solid-state N M R is used to study proteins in phospholipid bilayers. Although structural information is available under favorable circumstances, most of the studies to date have focused on the dynamics of proteins in bilayers where overall reorientation is slow. Bacteriorhodopsin (Kinsey et al., 1981a,b, 1984; Keniry et al., 1984; Smith & Oldfield, 1984; Rice et al., 1981; Schramm et al., 1981), filamentous phage coat proteins (Frey et al., 1983; Bogusky et al., 1985b; Colnago et al., 1986; Valentine et al., 1985; Leo et al., 1987), synthetic model peptides (Pauls et al., 1985; Mueller et al., 1986), and gramicidins (Datema et al., 1986) in phospholipid bilayers have all been studied in detail by solid-state NMR. Solution N M R methods are appropriate for relatively small membrane proteins solubilized in detergent micelles. Protein-micelle complexes undergo fairly rapid overall reorientation in solution, and as a result, high-resolution one- and two-dimensional N M R spectra of the protein can be obtained. Glucagon (Braun et al., 1981) and mellitin (Brown et al., 1982) were the initial model systems studied in micelles by high-resolution 'H NMR. Filamentous bacteriophage coat proteins in micelles have also been studied by solution N M R methods (Cross & Opella, 1979, 1980, 1981; Bogusky et al., 1985a,b; Hagen et al., 1979a,b; Dettman et al., 1982; Henry et al., 1986a,b; Wilson & Dahlquist, 1985). The coat protein of a filamentous bacteriophage is associated with the bacterial cell membrane during the viral life cycle (Webster & Lopez, 1985). The coat protein molecules from an infecting virus particle, as well as the newly synthesized coat protein molecules, insert into the host cell membrane (Smilowitz et al., 1972). Assembly of new virus particles occurs at the cell membrane, where the coat protein subunits aggregate to form a symmetrical outer shell around the viral DNA. The analysis of X-ray diffraction patterns from oriented fibers of filamentous bacteriophages suggests that there are two classes of these viruses, with fd and M 13 representative of class I and Pfl representative of class I1 (Marvin & Wachtel, 1975; Makowski, 1984). The overall features of the major coat proteins of both classes are quite similar. The coat proteins have large amounts of (Y helix in the virus particles, are small with 44-53 residues, and have a hydrophobic midsection flanked by basic and acidic hydrophilic terminal regions. The amino acid sequence of Pfl coat protein is shown in Figure 1. Although these proteins are insoluble in water and other solvents when separated from the virus particles, they are readily solubilized by a variety of detergents. These proteins adopt their membrane-bound conformations in the presence of detergents or lipids. The membrane-bound form has significantly less helical structure than the form that exists in the viral coat (Nozaki et al., 1976). This paper describes high-resolution 'H and I5N N M R studies of the Pfl coat protein in sodium dodecyl sulfate (SDS) micelles in solution. Even though the protein itself has a
molecular weight of 4600, the total molecular mass of the protein-micelle complex is about 28 000 daltons. The overall rotational correlation time of the complex is about 22 ns which results in short T2 relaxation times for the protons of the protein. The short T;s mean that the line widths in both oneand two-dimensional spectra are quite broad, making the N M R studies significantly more difficult than is typically the case for a protein of this size. Several methods were used to overcome these difficulties. In particular, I5N labeling made many one-dimensional "N NMR and two-dimensional 'H/ 15N N M R experiments feasible. The existence of a significant number of very slowly exchanging amide hydrogens is exploited to characterize the structure and dynamics of part of the hydrophobic midsection of the protein. The residues with the most slowly exchanging amide hydrogens are underlined in the sequence in Figure 1. MATERIALS AND METHODS Coat Protein Samples. Infection at early log phase of a 1-L rich medium culture of the host cells Pseudomonas aeruginosa strain K typically yields 100-150 mg of Pfl virus. The host cells are removed from the growth medium by low-speed centrifugation. The virus particles precipitate from the supernatant when it is made to be a 2% polyethylene glycol-0.5 M NaCl solution. The virus is pelleted by low-speed centrifugation. The pellet containing virus particles is resuspended in H 2 0 for 24 h and then repelleted. Virus purification is completed by layering the phage onto a KBr block gradient and centrifuging at 24000 rpm for 18 h in an S W 27 rotor. The virus-containing band on the gradient is removed and then dialyzed against a borate buffer at pH 8 with a small amount of ethylenediaminetetraacetic acid (EDTA) added prior to N M R sample preparation. 15N can be incorporated into the coat protein either uniformly or by amino acid type. Uniform 15N labeling is accomplished by using a minimal mineral growth medium supplemented with (15NH&S04 (Monsanto Research Corp.) as the sole nitrogen source. I5N is introduced into specific sites of the coat protein by growing the phage on a minimal growth medium supplemented with the I5N-enriched amino acid (Cambridge Isotope Labs) of interest. Scrambling of the 15N label via metabolic pathways to unintended backbone sites does not occur to a significant extent for the residues discussed in this paper. The 5-8 mM coat protein solutions are prepared as samples for 'H N M R studies by solubilizing 10-17 mg of intact phage with sodium dodecyl sulfate. Virus concentrations were determined spectrophotometrically by using an extinction coefficient of 2.07 cm2 mg-I at 270 nm (Day & Wiseman, 1976). Perdeuteriated sodium dodecyl sulfate (Merck Sharp & Dohme Isotopes) is added in a 2:l weight ratio of SDS to coat protein to a 10-mL solution of intact phage at 50 'C and neutral pH. Solubilization of the coat protein into SDS micelles is followed spectrophotometrically. After solubilization is complete, the solution pH is adjusted, and the sample is lyophilized. All pH measurements are made at 20 'C without correction for glass electrode deuterium isotope effects. The IH N M R samples are prepared by dissolving the lyophilized material in 0.4 mL of H 2 0 or D 2 0 in 5-mm NMR tubes. I5N N M R samples typically are 2 mL of a 2 mM coat protein solution solubilized in electrophoretic-grade SDS (Boehringer Mannheim Biochemicals) in 10-mm N M R tubes. The CD spectrum was obtained on a Jasco-Durram Model J10 instrument. N M R Techniques. Homonuclear J-correlated spectroscopy was performed by using the conventional two-dimensional 'H
BACTERIOPHAGE COAT PROTEIN
homonuclear correlation (COSY) experiment (Aue et al., 1976). The absolute-value COSY spectrum shown was recorded at 500 M H z in D 2 0 with weak coherent irradiation of the residual HDO resonance. A total of 2K data points were digitized during t 2 at each of 512 tl points. Zero filling in t l results in a 1024 X 1024 transformed data matrix with 4.8 Hz/point resolution in both dimensions. Phase-shifted sine bell window functions were applied in both dimensions prior to Fourier transformation. Proton spatial proximity was determined by using the two-dimensional NOE experiment (Jeener et al., 1979). Phase-sensitive 'H homonuclear Overhauser effect (NOESY) spectra were constructed following the method described by States et al. (1982). 500-MHz spectra were recorded in D 2 0 at 50 OC using a 250-ms mix time. A total of 2K data points were digitized at each of 512 tl points. Zero filling in tl results in a 1024 X 1024 transformed data matrix with 4.8 Hz/point resolution. Phase-shifted sine bell window functions were applied in both dimensions prior to Fourier transformation. All ' H N M R spectra are presented in unsymmetrized form. Two-dimensional IH/l5N chemical shift correlation spectra were obtained from both uniformly and specifically 15N-labeled protein samples in H 2 0 and in D 2 0 as described previously (Bogusky et al., 1985a). In the two-dimensional technique used, the detected 15N signal is phase modulated by the resonance frequency of the directly bonded amide proton. The spectrum correlates the chemical shifts of directly bonded amide 'H and I5N pairs without the effects of heteronuclear Jcoupling (Bax & Morris, 1981). Typically, 256 scans at each of 32 or 48 t l points at 1-ms increments were obtained with a 1-kHz I5N spectral window in t2. These sweep widths cover the entire amide proton and nitrogen spectral regions at 400 MHz. Heteronuclear 15N/'H N O E measurements on uniformly and specifically I5N-labeled protein samples were performed with a single I5N pulse and continuous broad-band proton irradiation. For direct comparison, single-pulse 15N spectra without N O E enhancement were acquired with proton decoupling gated on during acquisition only. Other amide I5N spectra were recorded by using the DEPT pulse sequence with proton decoupling during acquisition (Pegg et al., 1982a,b). The spectra were obtained on JEOL GX-400WB and GX500 N M R spectrometers. Two-dimensional data sets were transferred via direct Ethernet linkages or by magnetic tape to a Digital Equipment Corp. MicroVax I1 computer. All data sets were processed by using the "FTNMR" program obtained from D. Hare (Infinity Designs). RESULTS Pfl Coat Protein Has Stable Secondary Structure. Circular dichroism (CD) spectra are sensitive to the secondary structure of proteins. Figure 2 is a C D spectrum of Pfl coat protein in SDS micelles. This spectrum is very similar to those attributed to (Y helices in proteins (Greenfield & Fasman, 1969), and it indicates that Pfl coat protein in SDS micelles has secondary structure that is more than half a helix. The stability of secondary structures in proteins results, in part, from hydrogen bonds among peptide backbone sites. The resistance of peptide amide protons to chemical exchange with solvent protons (or deuterons) is highly indicative of stable secondary structure in proteins (Englander & Kallenbach, 1984). Dramatic retardations in the amide proton exchange rates of backbone sites involved in stable secondary structure compared to unstructured sites have been observed. Slowing factors greater than lo5 for amide proton exchange rates have been observed in a helices in proteins (Wand et al., 1986;
1375
VOL. 26, NO. 5 , 1987 I
I
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2: Circular dichroism (CD) spectrum of Pfl coat protein in 40 mM SDS at 20 OC. FIGURE
l
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3: Complete 'H NMR spectrum of Pfl coat protein in perdeuteriated SDS micelles in D20 at 75 "C and pH 4.4. This spectrum was obtained at 500 MHz with weak coherent irradiation of the residual HDO resonance. The labels correspond to (a) residual HDO, (b) buffer, (c and d) residual protons on SDS, and (e) overlapping resonances from SDS and protein methyl groups. FIGURE
Roder et al., 1985). Structure-induced chemical shift dispersion is also indicative of the presence of secondary and tertiary structure in a protein in solution. Figure 3 is the 'H N M R spectrum of Pfl coat protein in perdeuteriated SDS micelles in D20. Chemical shift dispersion among carbon- and nitrogen-bound protons as well as the presence of many amide resonances from slowly exchanging sites is observed. Pfl coat protein has two tyrosines and no other aromatic residues. The spectral region near 7 ppm shows resolution of the 2,6 ring proton resonances of the two Tyr residues. Some of the amide sites in the protein have protons which are very resistant to solvent exchange, since at least 11 amide protons remain unexchanged after 48 h in D,O at 75 O C and pH 4.4. A larger number of amide sites with inter-
1376
BIOCHEMISTRY
SCHIKSNIS ET AL.
I/
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One-dimensional 15Nand two-dimensional IH/IsN heteronuclear correlated NMR spectra of uniformly 15N-labeledPfl coat protein in H 2 0 and in D20at 75 OC and pH 4.4. (A) 15NNMR spectrum of Pfl coat protein in H 2 0 obtained with a DEPT pulse sequence. (B) Two-dimensional 'H/15N heteronuclear correlated spectrum. (C) Same as (A) except in DzO. (D) Same as (B) except in D20. FIGURE 5:
D.
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4: Downfield regions (6.2-9.2 ppm) of 'H NMR spectra of Pfl coat protein in D 2 0 obtained after the sample was subjected to various procedures that affect amide proton exchange. All samples started as powders of protein in SDS lyophilized from HzO. (A) This spectrum was obtained at 25 OC 3 h after resuspending a protein sample in D20at pH 4.4. (B) This spectrum was obtained at 50 OC approximately 20 min after raising the temperature of the same sample in (A). (C) This spectrum was obtained at 75 OC 2 days after raising the temperature of the sample used for (A) and (B). (D) This spectrum was obtained at 50 OC from a sample lyophilized from H20 at pH 9 and resuspended in DzOat pH 9 at 60 OC. The sample temperature was maintained at 60 OC for 30 s and then lowered to 0 OC, and the pH was lowered to 4.1 with DCI. The sample was then relyophilized and resuspended in D 2 0 at pH 4.3. FIGURE
mediate proton exchange rates also exists. Expansions of the spectral region containing the amide and aromatic resonances of 'H NMR spectra of Pfl coat protein obtained in D 2 0under several different conditions are shown in Figure 4. The spectrum in Figure 4A shows that approximately two-thirds of the amide protons remain unexchanged after 3 h in D20 at 25 "C and pH 4.4. Approximately one-third of the amide protons remain unexchanged after the temperature of the sample is raised to 50 O C for 20 min as seen in the spectrum in Figure 4B. Raising the temperature to 75 O C for several hours results in no additional exchange. The resolution among the amide proton resonances is quite good at the higher temperatures, and there is no evidence of changes in protein structure as a function of temperature. The spectrum in Figure 4 D was obtained after the pH of the sample was raised to 9; it demonstrates that all of the protein backbone sites in the micelle complex are fully accessible to the solvent, since all of the amide protons exchanged with solvent deuterons in less than 30 s. The 'H N M R data in Figures 3 and 4 indicate the presence of extremely stable secondary structure in the membranebound form of Pfl coat protein. The qualitative separation
of amide sites into those undergoing relatively rapid exchange and those that are highly resistant to exchange is demonstrated with better spectral resolution in one-dimensional I5N N M R spectra and two-dimensional lH/I5N heteronuclear correlated spectra obtained from 15N-labeledprotein samples. Uniformly I5N-labeledPfl coat protein in D20 yields I5N resonances only from those amide sites with bonded protons rather than deuterons in spectra obtained by polarization transfer, since the magnetization is transferred to the I5N nucleus from the directly bonded 'H nucleus. Therefore, I5N N M R spectra generated by polarization transfer can be used to monitor amide proton exchange. The one- and two-dimensional spectra in Figure 5B were obtained from a uniformly I5N-labeled protein sample which was maintained in D 2 0 at 75 OC and pH 4.4 for over 48 h. There are clearly 11 amide sites which remain unexchanged under these conditions, since there are 11 distinct resonances. The 11 backbone sites with slowly exchanging amide protons are the focus of the experiments described below. Assignment of the Slowly Exchanging Amide Sites. Although Pfl coat protein is relatively small, with only 46 residues as shown in Figure 1, the N M R relaxation properties of the protein are determined largely by the overall reorientation rate of the ca. 28 000-dalton protein-micelle complex (Makino et al., 1975). The experimental limitations imposed by the generally short T2's of the protein 'H resonances are particularly severe at the rigid peptide backbone sites involved in secondary structure. Two-dimensional lH homonuclear N M R spectra of Pfl coat protein exhibit little or no intensity in key spectral regions, especially those involving amide proton resonances. As a consequence, conventional systematic assignment procedures (Wuthrich et al., 1982) are of limited value. Several NMR techniques, in particular those involving 15N, were used to overcome the difficulties encountered in many two-dimensional 'H N M R experiments. An approach that combines data from IH/I5N heteronuclear chemical shift correlation spectra, ' H homonuclear J-correlated (COSY) spectra, and 'H homonuclear Overhauser effect (NOESY) spectra has been successful in assigning the 'H and 15N resonances from the amide sites with slowly exchanging protons to specific residues in the protein. Figure 5A shows the I5N N M R spectrum of uniformly labeled Pfl coat protein in H 2 0 . The narrow I5N lines com-
BACTERIOPHAGE COAT PROTEIN
A . HzO
ALLSITES
VOL. 2 6 , N O . 5 , 1987
A LA
ILE
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1377
VAL
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100
100
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FIGURE 6: Two-dimensional 'H/15N heteronuclear correlated spectra obtained on uniformly and specifically 15N-labeledcoat proteins in H20 and in D 2 0 as in Figure 5.
b i n d with the 20 ppm chemical shift dispersion result in nearly complete resolution of all of the backbone amide resonances, even in the one-dimensional spectrum. The two-dimensional IH/15N heteronuclear correlation spectrum in Figure 5B of uniformly I5N-labeled coat protein in H 2 0 demonstrates complete resolution of the resonances from all amide sites. In order to assign the 'H and 15N amide resonanm to amino acid types, lH/15N heteronuclear correlation spectra of specifically 15N-labeledprotein samples were obtained in H20and in DzO. This assignment method takes advantage of the high spectral resolution obtained in two dimensions, the ability to observe subspectra based on residue type, and the qualitative differences in amide proton exchange rates in the protein. The spectra obtained from uniformly 15N-labeled protein samples in H 2 0 and DzOare compared in Figure 5B and Figure 5D and in Figure 6A and Figure 6B. The two-dimensional heteronuclear correlation spectra of [ISN]Ala-, [I5N]Gly-, [I5N]Ile-, [15N]Leu-,and [lSN]Val-labeled protein samples obtained in HzO and in D 2 0 are compared in Figure 6. The data in Figure 6 are sufficient to assign 10 of the 11 'H and 15N resonances from the slowly exchanging amide sites to residue types. The one other amide site that undergoes very slow amide proton exchange is from Tyr-40. This resonance was assigned by specific chemical modification of Tyr-25 to differentiate the two sets of ring resonances from the two Tyr residues of the protein and by the observation of intraresidue 'H homonuclear NOES. The 2,6 ring resonances of Tyr-25 and Tyr-40 are clearly resolved while the corresponding 3,5 ring resonances overlap in the 'H N M R spectrum in Figure 3 and Figure 7A. The 3,5 ring positions of Tyr-25 can be specifically iodinated (Nave et al., 1981); this chemical modification of the protein results in changing the aromatic Tyr resonances to those shown in Figure 7B. In the spectrum of the modified protein, the resonances from the 3,5 ring protons of Tyr-25 are eliminated, and the 2,6 ring proton resonances collapse to a single line shifted downfield to about 7.8 ppm. The resonances from Tyr-40 at 6.7 and 6.9 ppm are unchanged. The resonances of the Tyr-40 ring were assigned to the respective 2,6- and 3,5-positions based on two-dimensional 'H N O E spectra and on finding that the resonance assigned to the 3,5-positions has a larger change in chemical shift upon titration of the phenol group than does the resonance assigned to the 2,6-positions. Figure 7C contains part of the two-dimensional ' H NOE spectrum of unlabeled and unmodified Pfl coat protein. Strong intraresidue cross-peaks from the Tyr-40 2,6 ring
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7: Tyrosine40assignment. (A) Unmodified Pfl coat protein. (B) Pfl coat protein with Tyr-25 iodinated at the 3.5-positions. (C) Phase-sensitive NOESY spectrum recorded at 500 MHz. The a,j3 to aromatic proton resonance region expansion shows the Tyr-40 2,6 ring proton (6.9 ppm) cross-peaks to the Tyr-40 a- and j3-proton resonances at 3.97 and 3.15, 3.10 ppm. The Tyr-40 amide proton resonance (8.86 ppm) shows strong NOE cross-peaks to the Tyr-40 a- and j3-proton resonances. FIGURE
proton resonance at 6.9 ppm to proton resonances at 3.97,3.10, and 3.15 ppm are present. This assigns the resonance at 3.97 ppm (c) to the a-proton of Tyr-40 and the resonances at 3.10 and 3.15 ppm (d) to the &protons of Tyr-40. The cross-peaks in the N O E spectrum from the a- and P-proton resonances (b and a) to the amide proton resonance at 8.86 ppm assigns this, the most downfield amide resonance that undergoes slow exchange with solvent, to Tyr-40. The corresponding 15N amide resonance at 92.9 ppm is assigned to Tyr-40 in the two-dimensional 'H/15N heteronuclear correlation spectrum in Figure 5D. The 'H resonances from the a and p sites of Tyr-25 were assigned in a similar manner. The 2,6 ring proton resonance from Tyr-25 gives rise to a cross-peak at 4.3 1 ppm,
1378
S C H I K S N I S ET A L .
BIOCHEMISTRY
Table I: Pfl Chemical Shifts" residue N-'H C,-'H I5N-H Leu-30 8.03 3.93 89.6 Val-3 1 8.17 3.45 90.7 Ile-32 7.92 3.51 91.7 Leu-33 8.20 3.93 92.5 Ala-34 8.35 93.6 Val-35 8.35 89.9 Ala-36 8.68 3.93 95.0 Gly-37 8.84 77.9 Leu-38 8.53 97.6 Ile-39 8.53 92.8 Tyr-40 8.86 3.97 92.9 OAmide and a-proton resonance chemical shifts are referenced internally to the Tyr 3,5 ring proton resonance at 6.70 ppm. Proton chemical shifts are given at 50 O C , pH 4.3. 15N chemical shifts are referenced to N-acetylglycine at 90.4 ppm and are given at 75 O C , pH
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FIGURE 9: Phase-sensitive NOESY spectrum recorded at 500 MHz of a 0.009 M solution of Pfl coat protein in DzO, pH 4.0, 50 OC. The mix time was 250 ms. The amide region of the protein showing NHi to NH,+l NOEs is plotted, and the sequential backbone walk is
43 . 6 3.8
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PPM FIGURE 8: Absolute-value COSY spectrum recorded at 500 MHz of a 0.009 M solution of Pfl coat protein in D20, pH 4.0,50OC. The fingerprint region (a-proton to amide proton J connectivities) of the
protein is shown. The assignments of the slowly exchanging amide proton and a-proton resonances are indicated. The digital resolution is 2.89 Hz/point after zero-filling. assigning this resonance to the Tyr-25 a-proton. Likewise, the resonances at 3.10 and 2.94 ppm were assigned to the /3-protons of Tyr-25. The data in Figures 6 and 7 assign all of the 'H and 15N resonances from 11 of the slowly exchanging amide sites in Pfl coat protein to residue types and one pair of amide 'H and 15Nresonances to Tyr-40. There is a contiguous 11-residue stretch of the protein sequence that contains Tyr-40 and one Gly, two Ala, two Ile, two Val, and three Leu residues, and that is the region encompassing residues 30-40. This part of the protein sequence is underlined in Figure 1. The contiguous arrangement of the residues with slowly exchanging amide protons is confirmed with the interresidue NOEs described in the next section. The 'H and 15Nchemical shifts and assignments for residues 30-40 are listed in Table I. The assignments to specific residues in Table I will be described in the next section. The assignments for the a-proton resonances for these 11 residues were made by using the two-dimensional 'H homonuclear COSY spectrum shown in Figure 8. This spectrum was obtained from a sample at 50 OC in D20. The displayed "fingerprint" region shows a-proton to amide proton J connectivities. Pfl coat protein has relatively little chemical shift dispersion among the a-proton resonances. Both relayed COSY and total correlated spectroscopy experiments were unsuccessful because the short proton T;s resulted in little or
no cross-peak intensity. Therefore, only 6 of the a-proton resonances are assigned and listed in Table I, even though all 11 of the amide proton resonances are assigned. The assignment of the Tyr-40 a-proton resonance was made on the basis of the NOESY spectrum in Figure 7C. Secondary Structure of the Pfl Hydrophobic Midsection. The amide resonance region of the two-dimensional NOESY spectrum of Pfl coat protein in D20is shown in Figure 9. Cross-peaks between amide protons indicate spatial proximity since magnetization is exchanged only over distances
