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SYSTEMATIC ANALYSIS OF CHEMICAL SHIFTS IN THE NUCLEAR MAGNETIC RESONANCE SPECTRA OF PEPTIDE CHAINS, I. GLYCINE-CONTAINING DIPEPTIDES* BY ASAo NAKAMURAt AND OLEG JARDETZKY4

DEPARTMENT OF PHARMACOLOGY, HARVARD MEDICAL SCHOOL Communicated by William N. Lipscomb, August 16, 1967 The interpretation of the nuclear magnetic resonance (NMR) spectra of proteins in structural terms is predicated on a quantitative knowledge of the spectral changes which accompany the polymerization of amino acids and their arrangement in specific secondary and tertiary structures. Despite some elegant work,'-4 the dec- ade which elapsed since the high-resolution NMR spectra of amino acids5' 6 and proteins7' 8 were first observed and qualitatively accounted for9 has left unanswered some of the most important questions, such as (1) the extent to which chemical shifts observed upon the incorporation of an amino acid into a peptide chain reflect the formation of a peptide bond at the N and the C terminal, respectively; (2) the effects of a given residue on the shifts of its nearest and next-nearest neighbors along the peptide chain; and (3) the side-chain dependence of the shifts observed on titration5' 6, 10 and helix-coil transition."-"5 It is the purpose of this and subsequent papers to show that the chemical shifts accompanying the incorporation of a given amino acid into a polypeptide chain can be systematized in terms of a few, relatively simple rules. The findings offer little hope for the use of NMR for the analysis of the primary sequence of large peptides, but underscore its usefulness in the study of secondary and tertiary structure. Experimental.-Most of the peptides were purchased and, if spectroscopically pure, were used without purification. All spectra were recorded on a Varian DP-60 high-resolution spectrometer, as previously described.'5 The measurements were made in DC1, D20, or NaOD solution at 30'C, unless otherwise specified. Sodium 2,2-dimethyl-2-silopentane- 5-sulfonate (DSS) was used as an internal and hexamethyl-disiloxane (HMS) as an external reference. All shifts are reported in counts per second (cps) or parts per million (ppm) from DSS. The concentration of each peptide was 0.05 M. The ionic form of each solute was identified by pD measurements using a glass electrode with a Beckman model G pH meter. The empirical correction pD = pH + 0.4 has been applied to all measurements. Solutions of peptide cations showed pD values of 0.9-1.0, those of zwitterions 5.4-6.0, and those of anions 12-13. In the case of peptides containing either more than one acidic or basic group, the pD value of the solution was adjusted so as to correspond to the fully ionized form. Results and Discussion.-Additivity of shifts: The chemical shifts of glycine- CH2 protons in peptides containing a glycine residue are summarized in Table 1. The data show several interesting features: (1) All chemical shifts of the CH2 group of glycine tend to cluster about six mean values, as follows: (a) glycyl- amino acid cations, 231.8 cps from DSS; (b) glycyl-amino acid zwitterions, 228.8 cps; (c) glycyl-amino acid anions, 199.0 cps; (d) aminoacyl-glycine cations, 242.5 cps; (e) aminoacyl-glycine zwitterions, 227.2 cps; and (f) aminoacyl-glycine anions, 225.0 cps. This immediately suggests that the shifts resulting from dif- 2212 Downloaded by guest on September 24, 2021 VOL. 58, 1967 CHEMISTRY: NAKAMURA AND JARDETZKY 2213

TABLE 1 CHEMICAL SIFrs OF GLYCIN-CH2 PROTONS IN PEPTIDES CONTAINING A GLYCINE RESIDUE Peptide Cation Zwitterion Anion Glycine 231.2 212.8 185.6 Glycyl-L-alanine 231.4 228.8 199.3 Glycyl-L-valine 233.7 232.6 201.3 Glycyl-L-leucine 232.3 229.9 199.4 Glycyl-L-isoleucine 233.5 231.7 200.8 Glycyl-L-methionine 232.9 230.7 201.2 Glycyl-L-serine 234.8 233.8 202.1 Glycyl-L-proline 239.8 236.8 206.2 Glycyl-L-aspartic acid 232.5 230. 9,a 228. 9b 199.4 Glycyl-L-glutamic acid 233.2 230.7,c 230.2d 200.1 Glycyl-L-lysine 233.0 233.5e 206.9,f 200.3 Glycyl-L-histidine 231.9 230.1,0 220.4h 196.3 Glycyl-L-tryptophan {227.41221.2 -* 189.0 Glycyl-L-phenylalanine 226.5 {227.3 192.9 Glycyl-f-alanine 226.9 226.4 196.6 L-alanylglycine 243.8 4231.3(224.8 226.1 L-valylglycine 244.8 {222.4 226.7 Lleucylglycine 244.0 2233.5 225.5 L-serylglycine 245.5 2265.42 227.4 4230.5 L-prolylglycine 244.8 (225.5 225.8 L-hydroxyprolylglycine 244.8 228.3 225.8 L-lysylglycine 243.8 228.2,i 226.3i 225.9 L-histidylglycine J240.2240.2 1229.7yt263221. 7 226.323.1 L-tryptophylglycine {2392t232.6~ ~ 12315.6412239.4 216.3 L-phenylalanylglycine 239.0 23136 212734 L-tyrosylglycine 239.9 221402 {219.4 fl-alanylglycine 241.0 226.0 225.2 Cps from DSS as an internal reference (60 Mc, cone. 0.05 M/1). (a) pD 3.61, (b) pD 6.94, (c) pD 3.79, (d) pD 6.85, (e) pD 3.59, Cf) pD 8.47, (g) pD 4.80, (h) pD 7.60, (i) pD 5.88, (j) pD 9.58, (k) pD 4.11, (1) pD 6.61. * Solubility is not adequate for measurement. ferences in the chemical nature of the nearest neighbor (at either the N- or the C- terminal) are an order of magnitude smaller than the changes in the chemical shift resulting from titration or from the formation of a peptide bond. (2) The chemical shifts of glycine in glycyl-amino acid cations and glycyl-amino acid zwitterions are almost identical (within 3 cps), and so are the shifts in aminoacyl-glycine zwitterions and aminoacyl-glycine anions, respectively (within 2.5 cps), indicating that the charge on the glycine-amino group dominates the shift, whereas the charge on the neighboring amino acid has only a small effect. (3) The shift of the CH2 peak in aminoacyl-glycine anions (226.0 cps) is approximately 40 cps (0.66 ppm) to lower fields as compared to the glycine anion (185.6 cps). Since the state of protonation of glycine is the same in the two cases, this large shift can be attributed directly to the formation of the peptide bond at the N-terminal. In contrast, (4) the shifts of the CH2 peak in glycyl-amino acid cations (231.8 cps) are almost identical to that in free glycine cation (231.2 cps). From this one may infer that formation of a peptide bond at the C-terminal makes only a small contribution to the over-all chemical Downloaded by guest on September 24, 2021 2214 CHEMISTRY: NAKAMURA AND JARDETZKY PRoc. N. A. S.

shift. Finally, (5) in a number of aminoacyl-glycines and some of the glycyl- amino acids, a chemical shift nonequivalence for the two methylene protons is observed, giving rise to an AB pattern in place of the usual glycine singlet. This has been previously observed by Mandel"6 for the middle residue of leucyl-glycylglycine and has been recently discussed by Morlino and Martin."7 Our findings are in agreement with theirs, except that because of the better resolution of our instru- ment we have been able to detect splitting in L-alanylglycine, L-serylglycine, and L-prolylglycine. Our interpretation of the phenomenon differs somewhat from theirs and will be discussed in the last section. The chemical shift differences apparent in Table 1 can be compared in a variety of ways. The most obvious and most informative comparison is between two species which differ by a single chemical change such as the glycine anion and the X-glycine anion, where the only chemical change is the substitution of the uncharged amino acid residue X- for a proton on the uncharged amino group of glycine. A comparison between the glycine zwitterion and the X-glycine zwitterion, on the other hand, reflects at least two chemical steps, i.e., the removal of the charge from the glycine-amino group, and the substitution of a positively charged residue X at the N-terminal. There are, in all, eight single-step comparisons which can be made on the data in Table 1. These are indicated diagrammatically in Figure 1. A single chemical shift term is assigned to each step. The remaining comparisons in Figure 1 are between species which differ by more than one chemical step. According to the above analysis, these over-all shifts may be expressed as a sum of terms, each reflecting one individual step. The analysis of the data in Table YCONHCH2COO- 1 and related data reported else-

X+CONHCHZCOOH4A!2-XCON c AnN 18 in the foregoing manner XtCONHCHZCOOH4* X+CONHCHCOO-X AEtN' , leadswhere16'to two important conclusions: XCONHCH2COO- (1) Chemical shifts which accom- ApN pany the incorporation of a given amino acid residue into a polypep- NH3+CH2COOH4--NH3+CH2C004-*Atc AtN NH2CH2COO- tide with a defined primary and ,APC secondary structure fall into two NHNH3+CH2CX3+CH2CONHX~nonoverlapping classes, when %AtC' ttNAM grouped according to their magni- , 'NH3+CHCONHX- 4 C2ONX EpC 3 tude. (2) The shifts are indeed additive to a very high degree of NH13 CHvZCONHY*approximation.* For convenience FIG. 1-Systematics of single-step chemical shifts in we shall refer to all shifts which glycine peptides. fall into the range of 0.3-1 ppm as first-order shifts and to those which fall into the range of 0.01-0.2 ppm as second-order shifts. It is noteworthy that first-order shifts resulting from changes in the primary or secondary structure are observed only for a-C protons, whereas second-order effects may be seen for both the ca-C and the side-chain protons.1'8 11, 15 Large chemical shifts affecting the side-chain protons may thus be taken as reflecting the tertiary structure of a polypeptide chain. The magnitude of the tertiary structure shifts for a-C protons Downloaded by guest on September 24, 2021 VOL. 58, 1967 CHEMISTRY: NAKAMURA AND JARDETZKY 2215

remains to be established, but because of the geometry of the polypeptide chain it is likely to be large only in exceptional circumstances. First-order shifts: Three first-order shifts are apparent in Table 1: (1) The shift due to titration of the N-terminal AtN is given by AtN -(A)-b(A)- -b(AX)± - S(AX)- = 0.45 ± 0.01 ppm, where b(A)± and b(A)- are chemical shift values of the a-CH protons of the free amino acids A in its zwitterionic and anionic form, and where b(AX)± and 5(AX) - denote the chemical shifts of the a-OCH protons of amino acid A which forms the N-terminal residue of a dipeptide. Note that AtN is a positive quantity denoting a downfield shift. (2) The C-terminal titration shift AtC is given by Ate B(A)+ -(A)± _ 5(AX)+ - b(AX)± = 0.3 4 0.01 ppm, where b(AX)+ and 5(AX)+ denote the chemical shifts of the a-CH protons of amino acid A which forms the C-terminal residue of a dipeptide in the cationic and zwitterionic form. Note that Ate is also positive, since the corresponding shift is also to lower fields. The two titration shifts, AtN and Atc, have been known since the initial NMR studies of amino acids and have been used by Scheinblatt'9 20 to identify the N- and C-terminal amino acids in peptides. The only noteworthy feature of the present data lies in the virtual independence of the titration shift from the nature of the neighboring amino acid at either the N- or C-terminal end. This is apparent from the small standard error of the mean for titration shifts in different peptides. (3) A third type of shift, the N-terminal peptide shift ApN is given by AIN =(XA) - (A)- - 0.66 0.01 ppm. In arriving at the value for AIN, the unusual chemical shifts seen with L-tryptophylglycine, L-phenylalanylglycine, and L-tyrosylglycine were omitted. For the sake of completeness, a fourth first-order shift should be added to those discussed above: (4) This is the helix-fornation shift Ah given by Ah = W(A)hel - (A)coi - -0.4 ppm, where b(A)helix and b(A),il represent the chemical shifts of the a-CH protons of amino acid A in the a-helical and random coil configuration.'5 Poly-L-glycine homopolymers are not known to form a-helices, and the helix-formation shift has not been measured in glycine-containing helical polypeptides. In view of the Downloaded by guest on September 24, 2021 2216 CHEMISTRY: NAKAMURA AND JARDETZKY PROC. N. A. S.

regularities observed in other polypeptides,131-5 however, Ah for glycine is not likely to fall outside the range to +0.3 to +0.6 ppm. The chemical shift of ain a-CH peak in the spectrum of a polypeptide sobs may thus be predicted by the equation bobs - a(A)± + Atc - AtN + APN + Ah. (1) In the case of glycine CH2 peaks in nonhelical polypeptides, the uncertain term A,, is not required, and chemical shifts may be predicted to within 0.1 ppm. Second-order shifts: A closer inspection of the data reveals several much smaller, but nevertheless discernible shifts. (1') The C-terminal peptide shift Apc =(AX)+-b(A)+ 0.00-0.02 ppm. The nature of the nearest-neighbor peptide may influence the chemical shift of a-CH protons. Depending on the side on which the neighboring residue is attached, two second-order shifts are defined (2') The N-terminal neighbor shift AnN 6(XA)---(YA)- 4t0.05-0.1 ppm. (3') The C-terminal neighbor shift AnC = (AX)+ - (AY)+ _ 0.0-0.2 ppm, where X and Y represent different amino acids. These two shifts are of particular interest because one might hope to utilize them for the identification of nearest neighbors, and hence for sequence analysis. Shifts of the order of 0.2 ppm, if subject to a systematic interpretation, would be quite adequate for this purpose, since they can now be measured with reasonable accuracy. The titration of nearest neighbors may also influence the magnitude of a-CH chemical shifts. Two second-order titration shifts are defined by (4') The N'-terminal titration shift =tNI S(XA) - (XA)- _ 0.05-0.1 ppm. (5') The C'-terminal titration shift =tch 5(AX)+ -(AX)± ~ 0.05-0.1 ppm. Thus, a better approximation for the chemical shift of a glycine residue in the interior of a peptide chain is given by Bobs = a(A)± + A - AttN + ApN + Ah + (Apt + AnN + AnC)- (2) Downloaded by guest on September 24, 2021 VOL. 58, 1967 CHEMISTRY: NAKAMURA AND JARDETZKY 2217

The validity of this approximation is TABLE 2 illustrated in a sample calculation COMPARISON OF OBSERVED AND CALCULATED LINE POSITION FOR given in Table 2. The data to be pre- GLYCINE-CONTAINING DIPEPTIDES sented in the second and third papers Calcu- Ob- of this series as well as still incomplete Gly (2) inResiduetriglycine (i) lated*4.11 served4.07 observations on other amino acids are Gly (2) in tetraglycine (i) 4.11 4.10 consistent with this notion of approxi- Gly (3)(2) in tetraglycinepentaglycine(-+-)(±-) 4.114.06 4.123.98 mate additivity of chemical shifts at- Gly (3) in pentaglycine (+) 4.06 4.00 tributable to different effects. Gly (4) in pentaglycine (+) 4.06 3.98 Chemical shift nonequivalence of the 3.55* &aicppm.fromAINequation= 0.45, (2),Atc using= 0.3,valuesApN =5(Gly)0.66 ppm,= glycine protons: This effect deserves ApC = 0,ALN = 0.05. further comment, inasmuch as two different views16' 17 have been put forth as an explanation-i.e., (a) the existence of preferred rotamers and (b) incomplete averaging of electric field gradients in the presence of free rotation. The latter effect has been predicted by Pople21 for all molecules containing an asymmetric carbon atom. If the observed chemical shift nonequivalence were solely the result of the incomplete averaging of the electric field gradients of the positive charge on the asymmetric carbon atom of the substituent in the presence of free rotation, one would expect the following: (a) A pronounced nonequivalence of the /3CH2 protons in all amino acids containing an asymmetric a-carbon, since the effect of such gradients should be greatest for the immediate neighbors of the a-carbon. This is generally not found.6 10 16 22 23 (b) A substantial effect of the positive charge of the neighboring amino acid on the average shift of the glycine protons, i.e., AtN' comparable in magnitude to the shift difference of the A and B protons in the glycine AB quartet. In fact AtN' is very much. smaller. (c) Complete temperature independence of the relative shift of the A and B protons. The temperature dependence of the relative shifts in L-phe-gly, L-tyr-gly, L-val-gly is shown in Figure 2. It is in good agreement 20 with the findings of Morlino and Martin17 and L-Ph.-Gly indicates that the contributions reflecting a 15 L- Tyr-Gly redistribution of rotamer populations are measurable. 10 Z-Vae-GIy Thus our conclusion is that the observed 4- nonequivalence reflects largely the restriction 5 of rotation about these bonds. This, however, does not necessarily signify the exist- 0 20 40 60 80 100 ence of one or a small number of preferred TEMPERATURE ('C) rotamers, but rather a range of probable and FIG. 2.-The temperature depend- a rotamers. ence of the relative shift of the glycine range of less probable protons in L-phe-gly, L-tyr-gly, L-val- The argument is diagrammatically repre- gly. sented in Figures 3 and 4. Figure 3 defines the two angles 4 and 46 corresponding to rotations about the Ca",,'CO and N-Ca bonds. Viewing the molecule along these bonds (which are almost parallel) in a direction from NH3+ to C02-, we find that the values of 4 allowed to the NH3+ and X groups, respectively, are not equal. This is indicated by the shading in Figures 4a and b. The energy barriers between the rotamers in which these two Downloaded by guest on September 24, 2021 2218 CHEMISTRY: NAKAMURA AND JARDETZKY PROC. N. A. S.

900 900

H-N 180 0 0 0CO 0

2700 2700 H/^ (a) NH; ROTATING ABOUT THE (b) X SIDE CHAIN ROTATING ABOUT THE 3CG AXIS AXIS C 0 900Ca-CO C'a-CO

H N C H KR' H-N 1800 0O- C=O H-N 1800 0 if C=O

FIGS. 3 and 4.-Diagra- 2700 2700 matic representation of (c)GLYCINE-H(I)ROTATING ABOUT THE (d)GLYCINE-H(2) ROTATING ABOUT allowed and forbidden N - Ca AXIS THE N-Ca AXIS rotamers in aminoacyl Forbidden regions are shaded; the view is along the C'a-CO and N-Ca axis in the glycines. NH3' to COO direction. groups fall into the unshaded areas are relatively low. On the other hand, barriers for rotation of each group through the shaded areas are relatively high. The angles 4,6 relatively accessible to each of the two CH2 protons are similarly defined in Figures 4c and d. Superposition of (c) or (d) on (a) and (b), respectively, shows the range of configurations over which each of the CH2 protons can be cis (with respect to the plane of the peptide bond) with either the amino group or with the side chain of its N-terminal neighbor. It is in these configurations that the direct effect of the neighbor will be largest. The extent of overlap is clearly different for the two protons of an aminoacyl-glycine, but much less so in the case of glycyl-amino acids, because the barriers for the rotation of an N-terminal glycine about the C-CO bond are relatively low. This accounts for the preponderance of chemical shift nonequivalence in the former. The primary effect of charge in this view is to further restrict the averaging by increasing the lifetime of all rotamers in which the positive and negative charges appear cis to each other. Only where there is a large contribution from a magneti- cally anisotropic side chain are the shifts apparent without additional restrictions. * This work has been supported by grants from the National Science Foundation (NSF GB- 4697) and the U.S. Public Health Service (5-K3-GM-15,379-08). t Present address: Central Research Laboratories, Ajinomoto Chemical Company, Kawasaki, Japan. t To whom all correspondence should be directed. Present address: Department of Biophysics and Phdrmacology, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey. 1 Kowalsky, A., J. Biol. Chem., 237, 1807 (1962). 2 Kowalsky, A., Biochemistry, 4, 2382 (1965). 3 McDonald, C. C., and W. D. Phillips, in Proceedings of the 2nd International Conference on Magnetic Resonance in Biological Systems (New York: Pergamon Press, in press). 4McDonald, C. C., and W. D. Phillips, J. Am. Chem. Soc., in press. 6 Takeda, M., and 0. Jardetzky, J. Chem. Phys., 26, 1346 (1957). 6 Jardetzky, O., and C. D. Jardetzky, J. Biol. Chem., 233, 383 (1958). 7 Saunders, M., A. Wishnia, and J. Kirkwood, J. Am. Chem. Soc., 79, 3289 (1957). 8 Saunders, M., and A. Wishnia, Ann. N.Y. Acad. Sci., 70, 870 (1958). 9 Jardetzky, O., and C. D. Jardetzky, J. Am. Chem. Soc., 79, 5322 (1957). Downloaded by guest on September 24, 2021 VOL. 58, 1967 CHEMISTRY: NAKAMURA AND JARDETZKY 2219

10 Bovey, F. A., and G. V. D. Tiers, J. Am. Chem. Soc., 81, 2870 (1959). 11 Bovey, F. A., G. V. D. Tiers, and G. Filipovich, J. Polymer Sci., 38, 73 (1959). 12 Glick, R. E., W. E. Stewart, and L. Mandelkern, Biochim. Biophys. Acta, 120, 302 (1966). 13 Stewart, W. E., L. Mandelkern, and R. E. Glick, Biochemistry, 6, 143 (1967). 14 Marlborough, D. I., K. G. Orell, and H. N. Rydon, Chem. Commun. (1965), p. 518. 15 Markley, J. L., D. H. Meadows, and 0. Jardetzky, J. Mol. Biol., 27, 25 (1967). 16 Mandel, M., J. Biol. Chem., 240, 1586 (1965). 7 Morlino, V. J., and R. B. Martin, J. Am. Chem. Soc., 89, 3107 (1967). 18 Nakamura, A., and 0. Jardetzky, submitted for publication. 19 Scheinblatt, M., J. Am. Chem. Soc., 87, 572 (1965). 2' Scheinblatt, M., J. Am. Chem. Soc., 88, 2845 (1966). 21 Pople, J. A., J. Mol. Phys., 1, 1 (1958). 22 Fujiwara, S., and Y. Arata, Bull. Chem. Soc. Japan, 37, 44 (1964). 23 Nakamura, A., J. Chem. Soc. Japan, 86, 780 (1965). Downloaded by guest on September 24, 2021