Application of heteronuclear NOESY and COSY two-dimensional NMR experiments to sequencing peptides

JOURNAL

OF MAGNETIC

RESONANCE

82,369-373

( 1989)

Application of Heteronuclear NOESY and COSY Two-Dimensional NMR Experiments to SequencingPeptides PETER L. RINALDI*

AND FREDERICK

J. SWIECINSKJ

Department of Chemistry, Knight Chemical Laboratory, The University ofAkron, Akron, Ohio 44325; and Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44104 Received August 15,1988

Two-dimensional NMR techniques have provided important methods for determining the structures of peptides. In particular, COSY and related experiments have been extremely useful in determining proton NMR resonance assignments and in assigning the structures of the amino acid fragments which make up the protein. It is possible to trace cross peaks which identify the geminal and vicinal coupling interactions between protons within a given amino acid unit in a typical peptide chain. The pattern of this cross-peak network is unique for each of the amino acids. Unfortunately, total peptide structures cannot be determined in this manner because the continuity of vicinal coupling interactions is disrupted by the presence of the carbonyl group in the peptide. Other methods such as COLOC ( I, 2)) HMBC (3)) and selective INEPT (4) have been proposed for establishing molecular connectivities across nonproton-bearing substituents such as carbonyl groups; however, these methods owe their success to long-range C-H coupling interactions. Because the range of these interactions is large (0- 10 Hz) it is not possible to optimize a single experiment for all of the coupling interactions present. We report the use of selective 13C NOES from protons on adjacent atoms in the context of a 13C{ ‘H} heteronuclear Overhauser enhancement spectroscopy (HOESY) (5, 6) experiment to identify the primary sequence of amino acids. For carbons lacking attached protons, virtually all of the dipole-dipole interactions responsible for the existence of an NOE arises from geminal protons. Once the proton resonances (specifically (Yand NH protons) have been assigned from a COSY spectrum, 13C carbonyl resonances can be assigned based on the presence of cross peaks to H-a resonances in the 13C{ ‘H } HOESY spectrum. Each carbonyl resonance will also exhibit a second cross peak to an NH resonance, permitting the identification of the next amino acid residue in the sequence. * To whom correspondence should be addressed at The University of Akron, Akron, Ohio 44325.

369

0022-2364/89 $3.00 CopyriehtQ 1989 by Academic AI1 rights of rqwcduction

Press, Inc. in any form reserved.

370

NOTES

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S ( 1)

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as a model to illustrate the application of the methodology. In addition to illustrating its utility in sequencing peptides, the HOESY spectrum of 1 also provides definitive 13Ccarbonyl resonance assignments, thereby resolving conflicting assignments which have been reported ( 7,s). The COSY spectrum of 1 is presented in Fig. 1, illustrating the NH and H-cw resonance assignments and some of the vicinal connectivities. From this spectrum we have obtained all of the proton resonance assignments. This low-molecular-weight decapeptide exhibits twofold rotational symmetry; therefore signals from only five unique amino acid fragments are observed. Characteristic patterns for each type of amino acid residue are observed; for example, valine exhibits the NH-H-a-H-&Hy (methyl) connectivity pattern. The 75 MHz i3C NOES for the carbonyl carbons were measured at 50°C using a 100 mg sample of 1 in 2.5 ml of DMSO-d6 contained in a 10 mm tube. The results of this experiment are summarized in Table 1; all carbonyl NOES fell in the range of 1.2-l .3. Despite these small NOES, a 13C{ ‘H } HOESY spectrum of 1 can be obtained as illustrated in Fig. 2. The 2D spectrum was collected at 50°C with the following f2 parameters: 350 Hz spectral window (centered about the carbonyl region), 256 points, 18 and 39 PS 13C and ‘H 90” pulse widths, respectively; 256 transients were averaged for each of 128 FIDs, with the evolution time incremented to provide a 2 140 Hz spectral window in the f, dimension. The phase cycling was adjusted to provide a phase-sensitive spectrum according to the method of States et al. ( 9). The optimum mixing time of 0.5 s was determined by looking for the most intense carbony1 resonances from a series of HOESY spectra in which the evolution time was fixed at 0 s and the mixing time was incremented. A hypercomplex FT was performed on a 5 12 X 256 data table without any weighting of the raw data.

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FIG. 1. Phase-sensitive double-quantum-filtered COSY 2D NMR spectrum of 35 mM 1 in DMSO-d,, obtained at 300 MHz and 50°C. Connectivities between NH and CH, resonances are outlined providing these resonance assignments. Also iBustrated are the connectivities between the remaining protons of the valine fragment, illustrating its characteristic COSY pattern.

The carbonyl farthest downfield (172.0 ppm) exhibits a cross peak to H-a of leutine, identifying it as the carbonyl of the leucine fragment. This carbon also exhibits a cross peak to the N-H of phenylalanine, identifying the next amino acid in the TABLE 1 13CNMR Data for Carbonyls of 1 Cross peaks in HOESY 13Cshift

Carbonyl assignment

172.0 171.2 171.1 170.6 170.2

Phe Val Om Pro

LAXI

NOE 1.3 1.2 1.2 1.3 1.2

‘X Leu (4.59) Phe (4.40) Val(4.44) om (4.79) Pro (4.34)

NH Phe (8.97) Om (8.56) Leu (8.29) Val(7.21)

372

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FIG. 2. Phase-sensitive 13C{ ‘H} HOESY spectrum of 35 mM 1 in DMSO-$ obtained at 75 MHz and 50°C. Exact experimental conditions are described in the text. Cross peaks are observed between each “Ccarbonyl resonance and a single ‘H resonance in the CH, region, permitting the assignment of the “C signal to a specific amino acid fragment. With the exception of Phe carbonyl ( 17 1.2 ppm) which is bound to the nitrogen of Pro, each “C resonance also exhibits a cross peak to an NH proton resonance, allowing identification of the adjacent amino acid fragment,

chain. With the exception of phenylalanine, all the other amino acid carbonyls exhibit the same sort of pattern. The phenylalanine carbonyl exhibits only a single cross peak to H-CY.The absence of a cross peak between this carbon and a proton with a shift in the NH region identifies proline (without an NH) as the next amino acid in the sequence. The resulting chemical-shift assignments are summarized in Table 1. Note that independent 13C resonance assignments are not needed as these are derived from the HOESY spectrum. Also, note that the relative 13C carbonyl resonance assignments agree with those of Sogn et al. ( 7). Combined application of COSY and HOESY offers an excellent method for determining the primary structure of small peptides. It also provides a means of simultaneously assigning the carbonyl i3C resonances. We have performed i3C HOESY experiments with a solution of 1 in DMSO at 4.7,7.05, and 9.4 T. From these experi-

NOTES

373

ments we conclude that the optimum field strengths for obtaining HOESY spectra appear to be 4.7-7.0 T. At higher field strengths chemical-shift anisotropy dominates the relaxation of carbonyl groups reducing the magnitude of the NOE and offsetting any sensitivity gain which might be expected. Therefore, in order to successfully apply this methodology at higher field where higher sensitivity and better spectral dispersion will be achieved, heteronuclear rotating-frame experiments must be employed, analogous to the CAMELSPIN/ROESY ( 10, I1 ) experiments used for proton homonuclear NOE. The onset of slower molecular motion and higher field strength also produces broader carbon lines (linewidth at half-height > 3 Hz for our sample) as well as smaller NOES. This degrades the sensitivity of experiments such as COLOC which rely on the ability to resolve long-range couplings. We have not been able to obtain COLOC spectra correlating a-CH and NH protons with the carbonyl resonances using the same Gramicidin S sample which produced the HOESY spectrum in Fig. 2. ACKNOWLEDGMENT This work was supported in part by the National Institutes of Health Grant RR-O 1657. REFERENCES 1. H. KESSLER, W. BERMEL,

ANDC. GRIESINGER, J. Am. Chem. Sot. 107,1083 (1985). 2. H. KESSLER, C. GRIESINGER, J. ZARB~CK AND H. R. Looser, J. Magn. Reson. 57,33 3. A. BAX AND M. F. SUMMERS, J. Am. Chem. Sot. IO&2093 (1986). 4. A. BAX, J. Magn. Reson. 57,3 14 (1984). 5. P. L. RINALDI, J. Am. Chem. Sot. 105,5167(1983). 6. C. YU ANDG. C. LEW, J. Am. Chem. Sot. 106,6533 (1984). 7. J. A. SOGN, L. C. CRAIG, AND W. A. GIBBONS, J. Am. Chem. Sot. %,3306 (1974). 8. D. W. URRY , in “The Enzymes of Biological Membranes” (A. Martonossi, Ed.), Vol.

1 (I 984).

1, Chap. 2, pp.

31-69, Plenum Press, 1976. 9. D. J. STATES, R. A. HABERKORN, AND D. J. RUBEN, .T. Mugn. 10. A. A. B~THNER-BY, R. L. STEPHENS, J. LEE, C. D. WARREN, Sot. 106,811(1984). 11. A. BAX AND D. G. DAVIS,

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