Nuclear Magnetic Resonance Spectrum of Lysine-Vasopressin in Aqueous Solution and Its Structural Implications

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 69, No. 8, pp , August 1972 Nuclear Magnetic Resonance Spectrum of Lysine-Vasopressin in Aqueous Solution and Its Structural Implications (proton assignments/dimethylsulfoxide/peptide/conformation/amino acids) P. H. VON DREELE*, A. I. BREWSTERt, J. DADOK$, H. A. SCHERAGA*, F. A. BOVEYt, M. F. FERGER*, AND V. DU VIGNEAUD* * Department of Chemistry, Cornell University, Ithaca, New York 14850; t Bell Telephone Laboratories, Murray Hill, New Jersey 07974; and I Department of Chemistry, Carnegie-Mellon University, Pittsburgh, Pennsylvania Contributed by H. A. Scheraga and V. du Vigneaud, May 1$, 1972 ABSTRACT The peaks in the proton NMR spectrum of lysine-vasopressin in aqueous solution at ph 3-5 were assigned to particular amino-acid residues by the use of the results of dilution studies and NH-C'H and CaH-C"H decoupling experiments. The conformation of lysinevasopressin in water differs from its conformation in dimethylsulfoxide. This paper is part of a series (1, 2) in which physical methods (primarily NMR) are used to obtain the structure of lysinevasopressin (LysVP) in solution, with a view toward the eventual elucidation of structure-biological activity relationships. In the present paper, a study of LysVP in aqueous media at ph 3-5 is reported, and a comparison is made between the peak assignments in aqueous solution and in dimethylsulfoxide. MATERIALS AND METHODS LysVP was a purified synthetic preparation (3, 4) that possessed about 250 U/mg of rat pressor activity. It has the following structure, in which the numbers indicate the positions of the individual amino-acid residues: H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH The half-cystine residues in positions 1 and 6 are referred to as Cys-1 and Cys-6, respectively. The proton NMR spectra were recorded on a Varian HR- 220 spectrometer, and on a 250-MHz spectrometer described elsewhere (5). The sample concentrations were M in D20 or H20 containing acetic acid, and in deuterated dimethylsulfoxide [U-2H]Me2S0. The chemical shifts in aqueous solution are downfield from an external standard of tetramethylsilane (Me4Si) in CCl4, and in [U-2H]Me2SO are downfield from Me4Si as an internal standard. In the dilution studies, t-butanol was added as an internal standard in some cases because Me4Si is not miscible with the solvent over the Abbreviations for amino-acid residues and protecting groups are in accordance with the IUPAC-IUB Tentative Rules [J. Biol. Chem. 241, 2491 (1966)]. All optically active amino acids are of the L-configuration. LysVP, lysine-vasopressin; J, coupling constant. Requests for reprints should be addressed to Dr. Scheraga at Cornell University. Pressor assays were performed on anesthetized male rats, as described in The Pharmacopeia of the United States of America (Easton, Pa.: Mack Publishing Co., 1970), 18th revision, p whole range of compositions studied. This procedure led to slightly different bulk magnetic susceptibilities at various dilutions, for which we corrected (correction <0.04 ppm) by alignment of the Tyr aromatic protons when aligning the spectra from the three dilution studies. Therefore, the absolute values of the chemical shifts at the intermediate compositions are not rigorously defined; however, those in the pure solvents, to which the conformational studies pertain, are defined as described above. The measured ph was corrected to pd, by use of the relation pd = ph when D20 was used (6). The spectra were obtained at ph about 5 to avoid (a) instability of the disulfide bond and (b) more rapid exchange of amide protons with those of water at higher ph. After the NMR measurements were completed, the LysVP solutions were bioassayed. No appreciable inactivation of the LysVP was detected. RESULTS AND DISCUSSION Assignments of peaks by the dilution method Coupling constants and temperature coefficients of the chemical shifts for the NH resonances in the NMR spectrum of LysVP in aqueous solution have been reported (7), but no peak assignments were made. Since the structural information contained in the NMR spectrum can be interpreted only when the peaks in the spectrum are correctly assigned to particular protons in the molecule, a careful peak assignment is essential in order to obtain the structure of LysVP 'in solution. In the past, this has been difficult to accomplish for polypeptides in aqueous media, since the CGH protons (which are coupled to NH protons) give rise to peaks that lie under a large HOH peak in H20 at 4-5 ppm; while the CaH peaks are observable in D20, the peaks for NH protons disappear because these are exchanged to ND. We have tried two approaches to the problem of peak assignment, both of which were successful. The first approach in assigning the NH peaks was to begin with a completely assigned proton NMR spectrum in another solvent, and to plot the chemical shifts of the NH peaks in mixtures of H20 and the other solvent as a function of the water content, thereby relating peaks observed in water to peaks observed in the other solvent. The reference solvent used in this case is [U-2H]Me2SO, since the peak assignments have already been made for LysVP in this medium (1, 2). The data were obtained from dilution studies at 200, beginning with 0.35 ml of 0.12 M LysVP in 0.25% (v/v) HOAc in H20 (ph = 5.0) and adding various aliquots of [U-2H]Me2SO until a concentration of 70 parts of [U-2H]Me2SO per 30 parts

2 2170 Chemistry: Von Dreele et al. Proc. Nat. Acad. Sci. USA 69 (1972) E Volume % HOH FIG. 1. The chemical shift of various peaks in the 220-M/Hz NMR spectrum of LysVP plotted against volume % of H20 in mixtures of [U-2H]Me2SO and 0.25% HOAc in H20 at 200. The symbols (0, A, and El) are the experimental points for three different experiments, aiid the lines are the proposed connections between the points. Filled symbols pertain to the peaks for the aromatic ring protons of the Phe and Tyr side chains. H20 (v/v) was reached, at which point the signal was too weak to measure. Similar dilutions, beginning in [U-2H]Me2SO and adding 0.25% HOAc in H20, gave data that covered the remainder of the composition range. The results of the dilution studies are shown in Fig. 1. Since the curves coincide in the overlap region, which is approached from opposite directions in the two series of dilutions, the system behaves in a thermodynamically reversible manner with no hysteresis effects. This approach will yield peak assignments provided that two conditions are satisfied, namely (a) that no highly cooperative change in conformation (which would result in an abrupt discontinuity in the curve) occurs as the solvent composition is changed, and (b) that several peaks do not appear at the same chemical shift at a given solvent composition. As can be seen in Fig. 1, condition (a) is satisfied for all NH protons (in the range of ppm), while condition (b) fails for the backbone NH protons (those in the range of ppm). By comparison of the data from the analogs 1-deamino-4- decarboxamido-lysvp, 4-decarboxamido-LysVYP, and 9- glycine methyl amide-lysvp with those from LysVP, we have assigned (2) the carboxamide peaks in [U-2H]Me2SO at 200 and found the order from high to low field to be Gln(cis-NH), Asn(cis-NH), Gly-NH2(cis-NH), Gly-NH2(trans-NH), Gln- (trans-nh), and Asn (trans-nh). These peak assignments are in agreement with those of Walter (8). From the data obtained in the dilution studies shown in Fig. 1, it is apparent that at 200 the carboxamide-nh proton peaks occur in the same order from high to low field in both [U-2H]Me2SO and H20. Therefore, at 200 the assignment of these peaks in H20, in order, from high to low field is the same as in [U-2H]Me2SO. From temperature studies in H20, we found that this peak order is the same at 200 and at 300. Therefore, based on the data from the analog studies, the dilution studies, and the temperature studies, the carboxamide-nh protons of LysVP in H20 at 30 and ph 5 are assigned as follows: 6.44 ppm, Gln (cis-nh); 6.49 ppm, Asn (cis-nh); 6.65 ppm, Gly-NH2 (cis-nh); 7.03 ppm, Gly-NH2 (trans-nh); 7.08 ppm, Gin (trans-nh); 7.18 ppm, Asn (trans-nh). These values are obtained from a spectrum of LysVP in water. They do not correspond to the numbers obtained if one attempts to use the scale on the left-hand ordinate of Fig. 1, which is referenced to internal Me4Si in [U-2H]Me2SO, to read the points on the right-hand ordinate, which is referenced to external Me4Si in CC14. The uncertainties in the overlap regions of Fig. 1 for the backbone NH protons were resolved by trying to preserve similar slopes for the various curves in the overlap regions; this procedure yielded the tentative curves shown in Fig. 1. The order for the backbone NH peaks (from high to low field) in [U-2H]Me2SO at 200 is 8.05 ppm, Asn; 8.15 ppm, Gly (triplet NH); 8.28 ppm, Gln and Lys (overlapping); 8.50 ppm, Cys-6; 8.59 ppm, Tyr (broad NH) and 8.86 ppm, Phe. These data lead to peak assignments for LysVP in aqueous solution at 200 from high to low field of Phe, Cys-6, Gln or Lys (for the NH having the smaller J of the two NH at this 5), Asn, Gly (triplet), Gln or Lys, and Tyr (broad peak) (see Table 1 for 6 values at 300). Assignment of peaks by the spin-decoupling method The second approach to peak assignment involves spindecoupling the NH from the CaH protons, and the C'H from the COH2 protons, and then matching the chemical shifts and splitting patterns of the COH2 protons to similar data that have been reported for each amino-acid residue in small peptides. The published data on the chemical shifts of the COH2 protons in water (9) (which are referenced to internal sodium 2,2- dimethyl-2-silapentane-5-sulfonate) and the chemical shifts of the COH2 protons in [U-2H]Me2SO (1) indicate that, to a first approximation, the relative positions of the chemical shifts of the COH2 protons are the same in both solvents for the aminoacids residues in LysVP. The spin-decoupling approach to identification of peaks in aqueous media is the most direct; however, it has not succeeded in the past because of instrumental difficulties arising when an attempt is made to irradiate the C'H protons located under the large HOH peak while decoupling is observed in the NH region. We have solved this problem in a manner explained elsewhere (Dadok, J., Von Dreele, P. H. & Scheraga, H. A., Chemical Commun., to be submitted), and the observed NH-CaH decouplings for LysVP in H20 are shown in Fig. 2A and Table 1. The positions of the C'H peaks (and their coupled NH peaks) were located by a blind-search irradiation under the H20 peak. The C'H peaks located in this manner were subsequently observed at the same positions when the spectrum was obtained in D20 (pd = 4.8). The Ca-CO decouplings (in D20) are shown in Fig. 2B. The coupling of the proton peaks at 4.39 ppm and 2.48 ppm (Asn) was observed both in H20 and in D20. The invariance of the COH peaks in H20 and D20, and also the coupling of the 4.39 ppm and

3 Proc. Nat. Acad. Sci. USA 69 (1972) NMR Spectrum of Lys-Vasopressin 2171 A 1020 Hz 1097 r Chemical Shift (ppm) FIG. 2. Spin-decoupling of the 250-MHz NMR spectrum of LysVP at 300 (A) in H20 at ph = 4.6 and (B) in D20 at pd = 4.8. The lowest spectrum in A and B is the undecoupled one obtained by irradiating off-resonance from all proton frequencies. The upper spectra show the various decouplings observed when irradiating at the frequency shown by each trace. Chemical Shift (ppm) 2.48 ppm peaks in both solvents, suggest that there is no significant difference in the conformation of LysVP in H20 and D20. Having established the decoupling relationships of the peaks, we assigned the peaks to particular amino-acid residues in the following way. The NH triplet at 8.01 ppm corresponds to the amino acid having two C'H protons, so that it and the decoupling-related C'H peak at 3.50 ppm were assigned to glycine. The C'H peak at 3.70 ppm, which is coupled to an NH at 7.88 ppm, is a one-proton peak that is coupled to the equivalent C'6H2 protons at 1.66 ppm. Although the COH2 resonances of Pro and Gln both occur in this region, the former does not have an NH peak. Therefore, the peaks at 1.66 ppm, 3.70 ppm, and 7.88 ppm were assigned to the CGH2, CaH, and NH protons, respectively, of Gln. The C"H peak at 4.07 ppm arises from two C'H groups, and is coupled to nonequivalent COH2 groups at 1.52 and 1.86 ppm (the Gln or Pro region) and at 2.92 and 2.56 ppm. Only one NH (7.69 ppm) can be spindecoupled from the C'H protons at this frequency; hence, one of the C'H protons is either Cys-1 or Pro, because neither of these residues has a peptide NH proton. Since the Gln peaks have already been assigned, and since the C'OH2 protons of Cys are generally between 2.4 and 3.2 ppm, the C$H2 resonances at 1.52 and 1.86 ppm and one C'H resonance at 4.07 ppm are those of Pro. The C6H2 protons of Lys are generally the highest-field C"H2 resonances of all the amino-acid residues in this molecule; therefore, the C~H2 resonances at 1.40 ppm and the related CcH resonance at 3.87 ppm and the NH resonance at 8.18 ppm were assigned to Lys. The 2.56 ppm peak is coupled to the 1.30 ppm peak, and they correspond to the e-ch2 and a-ch2 protons of Lys, respectively. The remaining amino acids that must be assigned are those with an amide, an aromatic ring, or a heteroatom at the y position (Asn, Phe, Tyr, Cys-6, and Cys-1). The CIH2 peaks

4 2172 Chemistry: Von Dreele et al. Proc. Nat. Acad. Sci. USA 69 (1972) TABLE 1. Peak assignments for backbone NH protons in the NMR spectrum of LysVP in water NH CaH CHH2 Assignment Assignment from NH v* a p* a v* a from dilution Type Hz ppm Hz ppm Hz ppm decoupling Phe Doublet t [ Phe L1020J Cys-6 Doublet ?$ Cys (Lys-Gln). Doublet (equiv) 1.66 Gln (small J) 415 Asn Doublet [ (equiv) 2.48 Asn L1097J 620 Gly Triplet None Gly (Lys-Gln) Doublet Lys Tyr (ph 4.6) Tyr broad (ph 3.2) [ Tyr doublet L1074J 4.29 None None Pro * Downfield from an external standard (Me4Si/CCl4) at 250 MHz, 30, and ph = 4.6. t Decoupling arrows point from the irradiating frequency to the frequency where a change is observed. $ The Jas of this proton is small, which makes detection of decoupling difficult. The appearance of a beat-frequency at 2120 Hz when irradiating at 1060 Hz made observation of NH-CaH decoupling difficult, hence, NH-CaH and CaH-CPH decouplings were not done at precisely the same frequency. The other proton could not be located. The broad peak at 3.05 ppm, which could not be decoupled from other peaks in the spectrum, may correspond to Tyr and/or Cys-1 protons. of these residues occur between 2.4 and 3.2 ppm. The C6H2 proton resonances of Asn in the LysVP precursors were found (1) to be fairly equivalent, and to occur on the upfield side of 'i ". 107.A I Chemical Shift (ppm) FIG. 3. (A) The backbone NH portion of the 250-MHz spectrum of LysVP in H20 at ph 3.2 and 300. The traces above the spectrum are a decoupled (1074 Hz) and undecoupled (4074 Hz) spectrum obtained by scanning very slowly with a large time constant to filter the beat occurring at twice the irradiation frequency. (B) A transfer of saturation experiment on the peaks shown in (C) at ph 4.6 and 300, irradiating first at 4090 Hz (off-resonance), then at 1097 Hz (the H20 peak), and recording the traces on top of each other. (C) The backbone NH portion of the 250-MHz spectrum of LysVP in H20 at ph 4.6 and 300. this region of the spectrum. As shown in Table 1, the 2.48 ppm peak is coupled to the 4.39 peak, which is coupled to the 7.90 peak. Therefore, the peak at 2.48, 4.39, and 7.90 ppm were assigned to C"H2, C'H, and NH, respectively, of Asn. The broad NH peak in the spectrum (at 8.48 ppm) at ph 4.6 (see Fig. 3C) was assigned to Tyr since, in LysVP, this amide is adjacent to the terminal amino group, which promotes the rate of exchange of the adjacent amide group at this ph (see next section). At ph 4.6 the NH is exchanging rapidly enough to eliminate the NH-CaH coupling, so that the C'H peak of Tyr cannot be located by spin-decoupling. When the ph was lowered to 3.2, the rate of exchange was reduced and the broad peak became a doublet at 8.50 ppm (Table 1 and Fig. 3A and 3C). This doublet is coupled to a CGH proton appearing near 4.29 ppm (Fig. 3A) that when irradiated appeared to give some change at 2.52 ppm (Fig. 2B). Therefore, these peaks were assigned to Tyr. The remaining two NH peaks belong to Phe and Cys-6, and are related by decoupling to the following peaks: 7.69 ppm (NH) to 4.07 ppm (C'H) to 2.92 and 2.56 ppm (COH2), and 7.74 ppm (NH) to 4.48 ppm (C'H) to 2.80 and 2.50 ppm (ClOH2). In [U-2H]Me2SO, the downfield C'H was assigned to Cys-6, in part because of an expected downfield shift due to the S-S group, and in part because comparison with the oxytocin spectrum (10) (with Ile replacing Phe) shows a C'H peak at the same downfield position, while the distinctive CaH of Ile is upfield. If the same downfield shift of the C'H of Cys-6 is preserved in aqueous solution, then the C'H resonance at 4.48 ppm is that of Cys-6 and the C'H resonance at 4.07 ppm is that of Phe. We now have two independent methods for making peptide peak assignments in the NMR spectrum of LysVP in aqueous solution. The results of the two methods of peak assignment (Table 1) agree very well.

5 Proc. Nat. Acad. Sci. USA 69 (1972) Effect of an N-terminal amino group An NH3+ group, as in NH3+-CH2-CONH-CH3, exerts an inductive effect on the nearby backbone amide group, resulting in an increase in the base-catalyzed rate of exchange of the NH proton (11). In the above compound, the basecatalyzed exchange rate constant of the amide (koh) is 120 times larger than that of CHr-CONH-CH3 (Me2CONH). A nearby peptide group also exerts an effect; however, it is usually much smaller. For example, CHrCONH-CHrCONH- CH3, which has an acetyl-nh moiety rather than an NH3+ near the CONH-CH3, has a koh for the CONH-CH3 group only 4 times larger than that of Me2CONH (11). Similarly, in glycyl-glycine, koh for the backbone NH nearest the C- terminus is 150 times that of Me2CONH, while in triglycine koh for the NH of the middle residue is 1300 times faster (12, 13). Since LysVP has a free NH3+ group at its N-terminus, the NH of Tyr should exchange faster than that of any of the other residues in the compound. The method of transfer of saturation (14) proved to be useful in identifying rapidly exchanging protons. If the rate of exchange of an exchangeable NH proton with water exceeds the rate of relaxation of the NH proton, then on irradiation of the water peak, the area of the peak corresponding to the NH proton will decrease because of the transfer of saturated protons from the water. This experiment was performed by a trace-superposition procedure that eliminates the need for peak integration, the decrease in area being visually observable. The observed backbone NH spectrum, obtained by irradiating first at 4090 Hz (off-resonance frequency) to avoid re-phasing the lock signal, and then at 1097 Hz (H20 resonance frequency) and recording the two spectra on top of each other, is shown in Fig. 3B. The broad NH peak at 8.48 ppm, which is present when the water peak is not irradiated (Fig. 3C), decreases in area when it is irradiated; hence, this peak corresponds to the rapidly exchanging backbone NH proton of the amino-acid residue in the 2 position-tyr. In this compound, a CGH peak also happens to be at the same chemical shift as the water peak, so that there is an additional change in Fig. 3B-that from decoupling the NH peak (Asn) at 7.90 ppm. This change alters the shape of the peak at 7.90 ppm, but does not change its area and, therefore, is readily distinguishable from a transfer of saturation. Further indication that the Tyr-NH peak is exchange broadened was obtained when the ph was lowered from 4.6 to 3.2 and the broad peak was observed to sharpen to a doublet (Fig. 3A). In triglycine, the onset of NH-CaH coupling in the amino-acid residue in the second position from the N-terminus was observed (13) when the ph was changed from 5.6 to 4.3, and was shown to be due to a slowing of the rate of NH proton exchange. This parallel onset of NH-CaH coupling in both triglycine and LysVP in the same ph range is another indication that the broadening is an exchange effect. The presence of the terminal NH3+ group in LysVP will promote a ph-dependent rapid exchange of the NH proton of the amino-acid residue in the second position which affects the NMR Spectrum of Lys-Vasopressin 2173 NMR studies in two ways: (i) the concommitant peak broadening can be used to assign the NH peak of the aminoacid residue in the second position from the N-terminus, e.g., Tyr in LysVP; (ii) the results of H-D exchange studies, which show a rapidly exchanging Tyr NH, do not necessarily indicate the absence of a hydrogen bond involving this NH. Comparison of the NMR spectra of LysVP in dimethylsulfoxide and in water The difference in the NMR spectra of the aromatic rings of Phe and Tyr of LysVP in water, where the rings are stacked (15), and in [U-2H]Me2SO, where they are not (2), is one indication that two different conformations are adopted by this molecule in the two different solvents. The observations in mixed solvents further illustrate this difference. As can be seen in Fig. 1, the Phe backbone NH peak, which is the lowestfield peptide peak in [U-2H]Me2SO, changes chemical shift gradually to become the highest-field peptide peak in H20. The other NH peaks also undergo similar changes in relative chemical shifts upon a change in solvent which suggests that LysVP has different conformations in water and dimethylsulfoxide. We are indebted to Dr. A. A. Bothner-By for helpful comments on the manuscript. This work was supported at Cornell by USPHS Grants AM and HL and by NSF Grant GB-28469X1, and was performed in part with the NMR Facility for Biomedical Research sponsored by National Institutes of Health grant RR P. H. V. -D. was an NIH predoctoral trainee, Von Dreele, P. H., Brewster, A. I., Scheraga, H. A., Ferger, M. F. & du Vigneaud, V. (1971) Proc. Nat. Acad. Sci. USA 68, Von Dreele, P. H., Brewster, A. I., Bovey, F. A., Scheraga, H. A., Ferger, M. F. & du Vigneaud, V. (1971) Proc. Nat. Acad. Sci. USA 68, du Vigneaud, V., Bartlett, M. F. & Johl, A. (1957) J. Amer. Chem. Soc. 79, Meienhofer, J. & du Vigneaud, V. (1960) J. Amer. Chem. Soc. 82, Dadok, J., Sprecher, R. F., Bothner-By, A. A. & Link, T., Abstracts of the 11th Experimental NMR Conference, Pittsburgh, Pa., April Glasoe, P. K. & Long, F. A. (1960) J. Phys. Chem. 64, Feeney, J., Roberts, G. C. K., Rockey, J. H. & Burgen, A. S. V. (1971) Nature New Biol. 232, Walter, R. 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