VOL. 14, 927-935 (1975)
BIOPOLYlIElZS
A 13C Spin-Lattice Relaxation Study of Dipeptides Containing Glycine and Proline: Mobility of the Cyclic Proline Side Chain E. T. FOSSEL, I8>6 Y>8%6
">@>a Y>8>6
~ > 8 > 6 y"i3>6 Y"B>6
y not available.
ing that the atoms lack conformational freedom. In the cyclic dipeptides the values of 7ring for Pro CB,Pro C', and Pro C8vary by up to a factor of two. In the linear dipeptides the values for these same atoms vary by a factor of three in cis Gly-cPro and by a factor of ten in trans Gly-cPro. These data indicate that the proline ring in cyclic dipeptides is less flexible than in the linear dipeptides. It is likely that the backbone rigidity of the cyclic dipeptides is transmitted to the conformationally interrelated atoms of the proline side chain. In comparing the proline-containing cyclic dipeptides, it can be seen that the introduction of the second proline as in cycZo(cPro-L-Pro), making it tricyclic compared to bicyclic cyclo(Gly-cPro), has the effect of slo%ing the motions of the carbons of the proline ring. The 7ring values for the 8, y, and 6 carbons in the cyclo(cPro-cPro) (Table 11) are identical within experimental error, as they are in cycZo(cPro-D-Pro), indicating that these three ring carbons are essentially moving as a unit. It is useful to compare qualitatively the motions of the 8, y, and 6 carbon atoms for each peptide studied. This information may then be compared to similar information from the prolinecontaining peptides mentioned earlier. Examination of these data (Table 111) shows that conformational equilibration in the proline ring is taking place in different ways, depending on molecular structures. In all of the linear peptides and in the cyclo decapeptide, gramicidin S, the order of motion falls into the group y >' 8 > 6. This is a result which would seem consistent with minimum restraint on the ring, since most motion is occurring a t the point most distant from the two anchor points (C" and N)of the ring. In these linear peptides and in gramicidin-S, backbone rigidity is lacking and conformational freedom in the proline ring is greatest. For two linear
NJIR STUDY OF IIIPEPTIDES
933
Fig. 2. Diagrammatic representation of cycZo(Gly-~Pro). The restriction of motion of the proline Cy is indicated.
peptides ~- Leu -~ -Pro -Glyand '~ polyproline 1118equilibrium between two ring conformers with the y carbon atom a t extreme positions have been observed. This observation is in accord with a theoretical study.20 Two slightly different orderings of ring carbon mobility exist in linear peptides. I n poly(Pro-Gly) and poly(G1y-Gly-Pro-Gly) the motions of the y and 0 are nearly equal, implying a restraint on the ring in this area. I n the cases of linear Pro-Gly and TRF, motions of 0 and 6 are nearly equal. I n the case of the three cyclic dipeptides studied, the rigidity of the peptide backbone is transmitted to the proline ring. As a reflection of this, a more limited number of conformations are available to the proline rings in cyclic dipeptides than in linear peptides. Only one of the two major conformations of the proline ring which represent nearly extreme positions of the y carbon are available for population in the three cyclic dipeptides studied. 2 1 , 2 2 A unique order of motion exists in cycZo(G1y-Pro). That is, 0 moves faster than y or 6 which are nearly equal. I n the cases of cyclo(L-Pro+ Pro) and cycZo(L-Pro-D-Pro), all of the carbon atoms move a t a similar rate, implying that these three carbon atoms may move as a unitary body in these more rigid '(tricyclic" molecules. A freezing out of the motion of the y carbon (Fig. 2) may mainly be responsible for the above result. This finding is consistent with theoretical studies.20s21 Besides the results on the proline ring, our data contain a t least one other piece of interesting information. This concerns the fact that since linear Gly-L-Pro exists as a mixture of cis and trans we have the opportunity t o examine the effect of this isomerism on molecular motions. With these two isomers and in linear L-Pro-Gly, N T , of Gly C" is considerably longer than NT1 of Pro C", indicating that the glycine portion of the molecule is undergoing more rapid motion than the proline portion. However, NT1 of the Pro C" is the same in both isomers.
934
FOSSEL, EASWARAN, AND BLOUT
CONCLUSIONS From application of the theory of spin-lattice relaxation in the case of protonated carbon atoms, we have obtained information concerning molecular motions of a molecule-both the tumbling motion and the rotation-vibrations of individual carbon atoms. We have applied this method to the study of rigid and partially rigid dipeptides containing proline and glycine. The molecular tumbling times, Teff, of the molecules were calculated from TIvalues, and the internal mobility correlation times, ~ ~ i , were calculated for the proline CB, Cr, and C6 atoms. The results show that modes of conformational equilibrium may be reflected in the ordering of the magnitude of motion of these three atoms. Comparison of our data on dipeptides to other data on other proline-containing peptides shows that placing proline rings in cyclic dipeptides changes the conformational equilibrium of the proline side chain from those of linear peptides. These changes may be both in the dominant conformational states present and in their rates of interconversion. An important difference is found between the conformational states present in linear peptides and the cyclic dipeptides. I n the cyclic dipeptides the motion of the y carbon of proline is slower than the motion of the y carbon in the linear peptides. This work was supported, in part, by U.S. Public Health Service Grants AM07300 and AM10794. The authors acknowledge with thanks the use of the XL-100-15 nmr instrument (provided by the National Science Foundation under Grant GP-32317). They also thank Mr. William Hull and Mr. Steve Patt for helpful discussions and suggestions, and for making their automated 21' program available. Dr. Vincent Madison and Dr. Charles M. Deber are thanked for many helpful suggestions and discussions. One of us (E. T. F.) thanks the Muscular Dystrophy Associations of American for a fellowship.
References 1. Deber, C. M., Fossel, E. T. & Blout, E. R. (1974) J. Amer. Chem. SOC.96,40154017, and references 2-13 listed therein. 2. Torchia, D. A. & Lyerla, J. R., Jr. (1974) Bwpolymers 13,97-114. 3. Deslauriers, R., Walter, R. & Smith, I. C. P. (1973) FEBS Letters 37.27-32. 4. Deslauriers, R., McGregor, W. H., Sarantakis, D. & Smith,'I. C. P. (1974) Biochemistry 13,3443-3448. 5. Allerhand, A. & Komoroski, R. A. (1973) J. Amer. Chem. SOC.95,8228-8231. 6. Deslauriers, R., Smith, I. C. P. & Walter, R. (1974) J . Amer. Chem. SOC.96,22892291. 7. Wallach, D. (1967) J . Chem. Phys. 47,5258-5268. 8. Woessner, D. E. (1962) J. Chem. Phys. 36,1-4. 9. Allerhand, A., Doddrell, D. & Komoroski, R. (1971) J. Chem. Phys. 55,189-198. 10. Doddrell, D., Glushko, V. & Allerhand, A. (1972) J. Chem. Phys. 56,3683-3689. 11. Glushko, V., Lawson, P. J. & Gurd, F. R. N. Gurd (1972) J. Biol. Chem. 247,31763185. 12. Schaefer, J. (1972) MacromOlecules5,427-440. 13. Allerhand, A. & Oldfield, E. (1973) Biochemistry 12,3428-3433. 14. Vold, R. L., Waugh, J. S., Klein, M. P. & Phelps, D. E. (1968) J. Chem. Phys. 48. 3831-3832.
, ~ ,
NMR STUDY OF DIPEPTIDES
935
15. Freeman, R. & Hill, H. D. W. (1971) J . Chem. Phys. 54,3367-3377. 16. Christl, M. &Roberts, J. D. (1972) J . Amer. Chem. Soe. 94,45654573. 17. Dorman, D. E. & Bovey, F. A. (1973) J . Org. Chem. 38,2379-2383. 18. Torchia, D. A. (1971) Macromolecules 4,440442. 19. Leung, Y. C. &Marsh, R. E. (1958) Acta Crystallog. 11,17-31. 20. Ramachandran, G. N., Lakshminayanan, A. V., Balasubramanian, R. & Tegoni, G. (1971) Biochem. Bwphys. Acta, 221,165-181. 21. Balasubramanian, R., Lakshminarayanan, A. V., Sabesan, M. N., Tegoni, G., Venkatesan, K. & Ramachandran, G. N. (1970) Znt. J . Protein Res. 3,25-33. 22. Young, P. E., Madison, V. & Blout, E. R. (1973) J . Amer. Chem. SOC.95,61426144.
Received January 31, 1975 Accepted March 7, 1975
BIOPOLYlIElZS
A 13C Spin-Lattice Relaxation Study of Dipeptides Containing Glycine and Proline: Mobility of the Cyclic Proline Side Chain E. T. FOSSEL, I8>6 Y>8%6
">@>a Y>8>6
~ > 8 > 6 y"i3>6 Y"B>6
y not available.
ing that the atoms lack conformational freedom. In the cyclic dipeptides the values of 7ring for Pro CB,Pro C', and Pro C8vary by up to a factor of two. In the linear dipeptides the values for these same atoms vary by a factor of three in cis Gly-cPro and by a factor of ten in trans Gly-cPro. These data indicate that the proline ring in cyclic dipeptides is less flexible than in the linear dipeptides. It is likely that the backbone rigidity of the cyclic dipeptides is transmitted to the conformationally interrelated atoms of the proline side chain. In comparing the proline-containing cyclic dipeptides, it can be seen that the introduction of the second proline as in cycZo(cPro-L-Pro), making it tricyclic compared to bicyclic cyclo(Gly-cPro), has the effect of slo%ing the motions of the carbons of the proline ring. The 7ring values for the 8, y, and 6 carbons in the cyclo(cPro-cPro) (Table 11) are identical within experimental error, as they are in cycZo(cPro-D-Pro), indicating that these three ring carbons are essentially moving as a unit. It is useful to compare qualitatively the motions of the 8, y, and 6 carbon atoms for each peptide studied. This information may then be compared to similar information from the prolinecontaining peptides mentioned earlier. Examination of these data (Table 111) shows that conformational equilibration in the proline ring is taking place in different ways, depending on molecular structures. In all of the linear peptides and in the cyclo decapeptide, gramicidin S, the order of motion falls into the group y >' 8 > 6. This is a result which would seem consistent with minimum restraint on the ring, since most motion is occurring a t the point most distant from the two anchor points (C" and N)of the ring. In these linear peptides and in gramicidin-S, backbone rigidity is lacking and conformational freedom in the proline ring is greatest. For two linear
NJIR STUDY OF IIIPEPTIDES
933
Fig. 2. Diagrammatic representation of cycZo(Gly-~Pro). The restriction of motion of the proline Cy is indicated.
peptides ~- Leu -~ -Pro -Glyand '~ polyproline 1118equilibrium between two ring conformers with the y carbon atom a t extreme positions have been observed. This observation is in accord with a theoretical study.20 Two slightly different orderings of ring carbon mobility exist in linear peptides. I n poly(Pro-Gly) and poly(G1y-Gly-Pro-Gly) the motions of the y and 0 are nearly equal, implying a restraint on the ring in this area. I n the cases of linear Pro-Gly and TRF, motions of 0 and 6 are nearly equal. I n the case of the three cyclic dipeptides studied, the rigidity of the peptide backbone is transmitted to the proline ring. As a reflection of this, a more limited number of conformations are available to the proline rings in cyclic dipeptides than in linear peptides. Only one of the two major conformations of the proline ring which represent nearly extreme positions of the y carbon are available for population in the three cyclic dipeptides studied. 2 1 , 2 2 A unique order of motion exists in cycZo(G1y-Pro). That is, 0 moves faster than y or 6 which are nearly equal. I n the cases of cyclo(L-Pro+ Pro) and cycZo(L-Pro-D-Pro), all of the carbon atoms move a t a similar rate, implying that these three carbon atoms may move as a unitary body in these more rigid '(tricyclic" molecules. A freezing out of the motion of the y carbon (Fig. 2) may mainly be responsible for the above result. This finding is consistent with theoretical studies.20s21 Besides the results on the proline ring, our data contain a t least one other piece of interesting information. This concerns the fact that since linear Gly-L-Pro exists as a mixture of cis and trans we have the opportunity t o examine the effect of this isomerism on molecular motions. With these two isomers and in linear L-Pro-Gly, N T , of Gly C" is considerably longer than NT1 of Pro C", indicating that the glycine portion of the molecule is undergoing more rapid motion than the proline portion. However, NT1 of the Pro C" is the same in both isomers.
934
FOSSEL, EASWARAN, AND BLOUT
CONCLUSIONS From application of the theory of spin-lattice relaxation in the case of protonated carbon atoms, we have obtained information concerning molecular motions of a molecule-both the tumbling motion and the rotation-vibrations of individual carbon atoms. We have applied this method to the study of rigid and partially rigid dipeptides containing proline and glycine. The molecular tumbling times, Teff, of the molecules were calculated from TIvalues, and the internal mobility correlation times, ~ ~ i , were calculated for the proline CB, Cr, and C6 atoms. The results show that modes of conformational equilibrium may be reflected in the ordering of the magnitude of motion of these three atoms. Comparison of our data on dipeptides to other data on other proline-containing peptides shows that placing proline rings in cyclic dipeptides changes the conformational equilibrium of the proline side chain from those of linear peptides. These changes may be both in the dominant conformational states present and in their rates of interconversion. An important difference is found between the conformational states present in linear peptides and the cyclic dipeptides. I n the cyclic dipeptides the motion of the y carbon of proline is slower than the motion of the y carbon in the linear peptides. This work was supported, in part, by U.S. Public Health Service Grants AM07300 and AM10794. The authors acknowledge with thanks the use of the XL-100-15 nmr instrument (provided by the National Science Foundation under Grant GP-32317). They also thank Mr. William Hull and Mr. Steve Patt for helpful discussions and suggestions, and for making their automated 21' program available. Dr. Vincent Madison and Dr. Charles M. Deber are thanked for many helpful suggestions and discussions. One of us (E. T. F.) thanks the Muscular Dystrophy Associations of American for a fellowship.
References 1. Deber, C. M., Fossel, E. T. & Blout, E. R. (1974) J. Amer. Chem. SOC.96,40154017, and references 2-13 listed therein. 2. Torchia, D. A. & Lyerla, J. R., Jr. (1974) Bwpolymers 13,97-114. 3. Deslauriers, R., Walter, R. & Smith, I. C. P. (1973) FEBS Letters 37.27-32. 4. Deslauriers, R., McGregor, W. H., Sarantakis, D. & Smith,'I. C. P. (1974) Biochemistry 13,3443-3448. 5. Allerhand, A. & Komoroski, R. A. (1973) J. Amer. Chem. SOC.95,8228-8231. 6. Deslauriers, R., Smith, I. C. P. & Walter, R. (1974) J . Amer. Chem. SOC.96,22892291. 7. Wallach, D. (1967) J . Chem. Phys. 47,5258-5268. 8. Woessner, D. E. (1962) J. Chem. Phys. 36,1-4. 9. Allerhand, A., Doddrell, D. & Komoroski, R. (1971) J. Chem. Phys. 55,189-198. 10. Doddrell, D., Glushko, V. & Allerhand, A. (1972) J. Chem. Phys. 56,3683-3689. 11. Glushko, V., Lawson, P. J. & Gurd, F. R. N. Gurd (1972) J. Biol. Chem. 247,31763185. 12. Schaefer, J. (1972) MacromOlecules5,427-440. 13. Allerhand, A. & Oldfield, E. (1973) Biochemistry 12,3428-3433. 14. Vold, R. L., Waugh, J. S., Klein, M. P. & Phelps, D. E. (1968) J. Chem. Phys. 48. 3831-3832.
, ~ ,
NMR STUDY OF DIPEPTIDES
935
15. Freeman, R. & Hill, H. D. W. (1971) J . Chem. Phys. 54,3367-3377. 16. Christl, M. &Roberts, J. D. (1972) J . Amer. Chem. Soe. 94,45654573. 17. Dorman, D. E. & Bovey, F. A. (1973) J . Org. Chem. 38,2379-2383. 18. Torchia, D. A. (1971) Macromolecules 4,440442. 19. Leung, Y. C. &Marsh, R. E. (1958) Acta Crystallog. 11,17-31. 20. Ramachandran, G. N., Lakshminayanan, A. V., Balasubramanian, R. & Tegoni, G. (1971) Biochem. Bwphys. Acta, 221,165-181. 21. Balasubramanian, R., Lakshminarayanan, A. V., Sabesan, M. N., Tegoni, G., Venkatesan, K. & Ramachandran, G. N. (1970) Znt. J . Protein Res. 3,25-33. 22. Young, P. E., Madison, V. & Blout, E. R. (1973) J . Amer. Chem. SOC.95,61426144.
Received January 31, 1975 Accepted March 7, 1975
