13C Nuclear Magnetic Relaxation of Poly(O-Glutamic Acid)

Polymer Journal, Vol. 11, No. 4, pp 299-306 (1979)

13 C Nuclear Magnetic Relaxation of Poly(o-glutamic acid) in Aqueous Solution

Toshifumi HIRA0KI and Kunio HIKICHI

Department of Polymer Science, Hokkaido University, Sapporo 060, Japan.

(Received July 20, 1978)

ABSTRACT: The molecular conformation and dynamics of poly(o-glutamic acid) in aqueous solution were studied by 13C nuclear magnetic resonance spectroscopy. Chemical

shift, spin-lattice relaxation time (T1), spin-spin relaxation time (T2), and the nuclear Overhauser enhancement (NOE) were measured as functions of pH at 300 K. Increasing pH resulted in upfield shifts of Ca and peptide C' carbons, reflecting the helix-to-coil

transition and downfield shifts of Cfi, C7, and Cs carbons, reflecting the ionization of the side-chain carboxyl group. Ti, T2, and NOE increased with increasing pH. The effective reorientational correlation time (-rerr) of Ca obtained from the combination of T1 and NOE was 2.8 nsec in the helix region. This indicates that Teff of Ca is determined by not only the overall motion of the molecule but also by appreciable local segmental motions of the backbone. When going to the coil, Terr of Ca decreases by a factor of 4.3 as a result of the

onset of rapid segmental motion. There is a progressive increase in T1 values of the side• chain carbons as going away from the backbone, suggesting that the end of the side-chain undergoes more a rapid internal reorientation than the others even in the helix state. The

peptide carbonyl carbons relaxes more slowly in D2O than in H2O, suggesting that the relaxation is contributed appreciably from the amide proton which is exchanged with

deuterium in D 2O solution. KEY WORDS Poly(o-glutamic acid) / Helix-Coil Transition / 13C NMR / Chemical Shift / Spin-Lattice Relaxation Time / Spin-Spin Relaxation Time / Nuclear Overhauser Enhancement / Correlation Time/

The conformational transition of a polypeptide relaxation time (T2), and the nuclear Overhauser in solution has been extensively studied using enhancement (NOE) are sensitive functions of the many physical techniques,1 including nuclear correlation time of reorientational motion of magnetic resonance (NMR) spectroscopy2 which internuclear vector. s-s Thus, these NMR param• can monitor individual atoms in a molecule. So eters are useful to estimate the correlation time far, the approach to the conformational study of molecular motion. of polypeptide by NMR was to observe the There are a few 13C nuclear magnetic relaxation change in the chemical shift caused by the confor• studies on homopolypeptides; poly(r-benzyl L• mational transition. The observed change in the glutamate),7'8 poly(L-lysine),9 poly(L-proline),10 and chemical shift was interpreted empirically by poly(L-hydroxyproline). 10 It is well known that reference to the results of other measurements, poly(o-glutamic acid) undergoes the coil-to-helix i.e., optical rotatory dispersion, circular dichroism, transition in an aqueous solution when the pH intrinsic viscosity, etc. is lowered. In the present work, we carried out In addition to chemical shifts, nuclear magnetic 13C NMR studies of poly(o-glutamic acid) in aque• relaxtion times provide useful information about ous solution in order to obtain information about the conformational transition of a molecule. the relationship between molecular motions and

Spin-lattice relaxation time (Ti), spin-spin the conformations. The chemical shift, Ti, T2,

299 T. HrRAOKI and K. HIKICHI and NOE of the backbone and side-chain carbons relative to the resonance of internal dioxane and were measured as functions of pH. corrected to tetramethylsilane (TMS) by the rela• tion of 0T!,IS=0dioxane+67.86 ppm.12 Spectral EXPERIMENTAL widths of 3002 Hz with SK data points and 1500Hz with 4 K data points were used. The T1 measure• The sodium salt of poly(o-glutamic acid)(PGA) ments were performed by the inversion recovery used in this work was prepared by alkaline hydroly• method13 ' 14 for protonated carbons and by the sis of poly (r-methyl o-glutamate) provided by the saturation recovery method15 ' 16 for nonprotonated

Ajinomoto Co. After exhaustive dialysis against carbons. T 2 was estimated from the measured distilled water and passing through a Chelex-100 line width Llv corrected for digital broadening,

(Bio-Rad) column to remove paramagnetic impuri• using the relation 1/T2 =irilv. The NOE was ties, PGA was lyophillized to a powder. The determined for protonated carbons by the com• degree of polymerization was determined to be parison with the intensities of fully decoupled and 260 from the intrinsic viscosity measured in 0.2-M gated decoupled spectra.17 ' 18 NaCl solution at pH 7.1 and at 298 K.11 PGA solutions were prepared at a residual con• RESULTS AND DISCUSSION centration of0.67 Min 99.8-% D2O obtained from Commissariat a l'Energie Atomique (CEA) and Chemical Shift in distilled and deionized H2O. Adjustments of The proton-decoupled 13C NMR spectra of pH were made with 1-N NaOD and DCl obtained PGA at pH 4.9 and 7.5 are shown in Figure 1. from Merck. The pH was measured on a Hitachi• The assignment of all peaks follows from that by Horiba M-7 pH meter equipped with a combina• Lyerla, et al. 19 It is apparent that the line widths tion micro-electrode. The pH values reported of all resonances are broader at pH 4.9 than at here are direct meter readings without correction pH 7.5. for any deuterium isotope effect. Since the Figure 2 shows the effect of pH on the chemical measured values of T1 of all the carbons were less shifts of PGA in the pH range of 4.8 to 10.1. than about 2 s ,no attempt was made to remove Since PGA strongly aggregates in the lower pH oxygen gas dissolved in the solution. region at concentrations studied here,20 measure• 13C NMR spectra were obtained at 15.04 MHz ments were made only above pH 4.8, at which using a JEOL FX-60Q Fourier transform spectro• pH PGA will be a partial helix in consideration of meter with a quadrature phase detector. All deuterium effect on the pH measurements.21 measurements were done at a temperature of 300K. Below pH 4.8 where white precipitates are formed The chemical shifts reported here were measured in the solution, it is very difficult to observe

c,, C'

dioxane

200 180 160 60 40 20 0 ppm from TMS Figure 1. Comparison of PGA spectra at two pH's at 300 K: a, pH 4.9, 14,000 scans; b, pH 7.5, 10,000 scans. Repetition time of 90° pulses is 1.5 s. Chemical shift scale in ppm from TMS.

300 Polymer J., Vol. 11, No. 4, 1979 13C Magnetic Relaxation of Poly(Glu)

resonances. 164 When pH increases, the a-carbon (C,) and the 16 2 peptide carbonyl carbon (C') resonances move

180 C6~ upfield by about 2 ppm, while the /3-carbon(Cp), the r-carbon(C7), and the side-chain carboxyl 176 carbon(Ca) resonances move downfield by com• 17 6 parable amounts. Keim, et al., have shown that c·~~ g_ 174 all 13C resonances of glutamic acid incorporated as a. ~172 central residue of a linear pentapeptide Gly-Gly• (/) :::;: Glu-Gly-Gly, not forming a helix, move down• I- field by 1-3 ppm with increasing pH. 22 13C NMR E 56c~~ studies on some homopolypeptides in aqueous and 54 non-aqueous :::: solutions indicated that upfield .c "l52 shifts of C,, and C' resonances are accompanied Ul 36 by the transition from helix to coii.7' 9 ' 19 , 23 , 24 0 u 34 Our results therefore indicate that the shifts of E C;~ a, C,, and C' resonances are due mainly to the .c 32 u opening of the hydrogen bonding of PGA, and 30 that the behavior of C~, C 7, and Ca resonances 26 reflect primarily the ionization of the carboxyl 26 group of the side chain. These results are in good agreement with those of Lyerla, et al., 19 24 4 5 6 7 8 9 10 11 pH except for the difference between D and L enan• Figure 2. pH Dependence of chemical shifts of PGA tiomers. at 300 K. -~--~--5

.-.--~---46

-~~---~100 --~------~--.,...., --11----,-1-----110 ----~---~ '--tt--+---~-120 '-ii------~130 ----~---''---'11 ------140 c°' c/3 1000

Figure 3. Inversion-recovery 13C spectra of Ca, Cp, and Cs carbons of PGA at pH 7.5 and at 300 K. Each spectrum is the result of 2,500 scans with a waiting time of 1.5 s. The delay time is shown at the right of each spectrum in milli-seconds.

Polymer J., Vol. 11, No. 4, 1979 301 T. HIRAOKI and K. HIKICHI

T1, T2 (Line Width), and NOE 25 Figure 3 shows the inversion-recovery Fourier c" 0 transform spectra of protonated carbons of 20 PGA. We show the pH dependence of T1 for ,::; c~ :r: each carbon in Figure 4. For C,, Cp, and C7 15 carbons, NT1 values are plotted, where N is the :S I number of protons directly bound to the carbon, 'O ,, 0 0 0 i 10 and for Ca and C' carbons, simply the T1 values oO X are plotted. In the region above pH 6, T1 values "'C C, _J of all carbons do not vary with pH, while with c·~ ' e g decreasing pH below 6 T 1 values decrease sharply. . . 0 The pH dependence of T1 values is very similar to 4 7 B 10 that of the chemical shift as shown in Figure 2. pH NOE and the line width are plotted against pH in Figure 6. pH Dependence of line widths of PGA at Figures 5 and 6, respectively. These results 300K: 0, Ca; e, Cp; x, Cr; 0, Ca; 0, C'. I means error bar.

10 suggest that the correlation time of tumbling mo• tion becomes longer in conjunction with the coil• to-helix transition. One of the dominant mechanisms for nuclear u relaxation of 13C nuclei is the dipole-dipole ""Ul interaction with neighboring protons, modulated by reorientational motions of macromolecules.5 ' 6 If the details of reorientation of 13C-1H inter• nuclear vectors are known, it is possible to present expressions of T1, T2, and NOE in terms of correlation times of reorientational motions. Here, we assume that a simplified model of iso• tropic motion with a single effective correlation O.O \'---~-~6-~7'------!-B-~~1"""0 ----c'11· time (,.ff) can describe the main feature of the pH nuclear relaxation. Then, T1, T2 , and NOE are Figure 4. pH Dependence of NT1 value for Ca, Cp, given by3 ' 5 and Cr carbons, and T 1 value for C' and Ca carbons 1 N fz2r/rH2 at 300 K. 1;=2{)•--r6 {J(wH-WC)

+31(Wo)+6J(w0 +wH)} (1)

3.0 1 N fz2r/rH 2 y;-=40· r 6 {J(wH-wc)+3J(w0 ) 2.5 +6J(w0 +wH)+4J(0)+6J(wH)} (2) w O 2.0 NOE=l +k. 6J(wH+w0 )-J(wH-wo) z ::F re J(wH-Wo)+3J(Wo)+6J(wH+wo) c. I 1.5 (3) and the spectral density is given by 1.0 4 6 2 (4) pH J(w)=2,err/O +w ,!rr) Figure 5. pH Dependence of NOE for protonated where ft is Planck's constant divided by 2rr, r O and carbons at 300 K: O, Ca; e, Cp; x, Cr. I means rH are the gyromagnetic ratios of 13C and 1H, error bar. respectively, r is the distance between 13C and 1H

302 Polymer J., Vol. 11, No. 4, 1979 13C Magnetic Relaxation of Poly(Glu) nuclei, and we and wH are the angular resonance Table I. Comparison of measured NOE and T2 frequencies of 13C and 1H nuclei, respectively. values of Ca carbon in the helix and the coil states with the values calculated using -r err Ca Carbon estimated from measured T1 at 300 K At pH 7.5 and at 300 K, a measured NOE of pH• 2.7 ±0.2 is maximal as shown in Figure 4, and is in the limit where NOE is no longer a sensitive func• 4.9 7.5 tion of the correlation time. This leads to two im• Measured Ti, ms 38 73 portant consequences: the nuclear relaxation of Measured -rerr, ns 2.8b 0.65 Ca is determined only by 13C-1H dipole-dipole Measured NOE 1.8 2.7 Calculated NOE interaction, and the extreme narrowing conditions 2.1 2.9 Measured T2, ms 16 37 are valid. Using eq 1 under extreme narrowing Calculated T2, ms 13 72 conditions, the T1 value of 73 ms measured at • Direct meter reading in D20 solution. pH 7.5 yields r 0 ff=0.65 nsec, assuming the C-H b This value was obtained from the combination of distance, r=l.08 A. T1 and NOE values. If we assume the polypeptide in the coil state as an equivalent rigid sphere, the correlation time width. of overall tumbling motion (re) is given by At pH 4.9 and at 300 K the observed value of Te=4r:r;r8/3kT (5) T1 is 38 msec and the NOE is 1.8. The combina•

tion of T1 and NOE values yields r 0 rr=2.8 nsec. where r; is the viscosity of the solvent and ro is the It turns out that under these conditions, the ap• radius of the sphere. The value of r O may be ap• proximation of the extreme narrowing is not proximated by the radius of gyration of a freely valid for Ca, At this pH, PGA is thought to be jointed chain of n unit of length l, l ,./n/6. Using in the helix region. Thus, it is reasonable to the degree of polymerization of 260 as n, l of 3.8 consider that the effective correlation time, longer 25 26 A, and r; of 10.5 mP for D20 at 300 K, we at pH 4.9 than at pH 7.5, is the consequence of obtain a correlation time of 17 nsec from eq 5. slower segmental motions of the molecules in It should be noted that this calculated value is the helix region. a lower limit, since the excluded volume effect is It has been supposed that a helical PGA not taken into account for ro and polymer chains molecule in solution behaves like a rigid prolate are subject to entanglements. The actual value of ellipsoid27 undergoing rotational diffusion charac• the correlation time for overall tumbling motion terized by two rotational diffusion constants. would be therefore greater than the calculated These are designated as D., and Db for rotation value of 17 nsec. The effective correlation time about major axis and rotation about an axis per• measured at pH 7.5 where the polypeptide is in pendicular to the major axis, respectively. The the coil state is significantly smaller than the dimensions of the ellipsoid, a (the major semi• calculated value for overall tumbling motion. axis) and b (the minor semi-axis), were each This suggests that the correlation time of Ca is assumed to be half the length of the helix and determined by local segmental motions of the to have an average radius in solution. Here, we backbone rather than by overall motion of the assume the diameter of the solvated helix to be molecule. It is found that the effective correla• 15 A, 27 and the length per peptide residue to be tion time obtained here is in good agreement with 1.5 A. For a highly prolate ellipsoid with an axial those of poly(r-benzyl L-glutamate)8 and of poly(L• ratio (p=b/a) much less than unity, D. and Db lysine)9 in the coil state at room temperature. are given by28 Using the effective correlation time obtained 3kT from T1 , we calculate NOE and T2 from 2 eq and D. = l6r:r;ab2 3. The results are shown in Table I. The calculat• ed NOE is consistent with the measured one. 3 Db kT 3 {21n (~)-1} (6) On the other hand, the calculated T2 is two times 16r:r;a p greater than the one estimated from the line And the spectral density is given b/

Polymer J., Vol. 11, No. 4, 1979 303 T. HIRAOKI and K. HrKrcm

2A-rA 2B-rn 2C-r0 indicate that Ci3 and Cr carbons of the side chain J(w) (7) l+w2-rA 2 + l+w2-rn2 + l+w2-r/ undergo internal reorientation. Such internal reorientation will be possible even in the helix where A=(3 cos2 0-1)2/4, -r:,: 1.=6Db, B=3 sin2 0· state, since the side chains of PGA are located cos2 0, -r;,1=Da+5Db, C=3 sin4 0/4, and -r;:/= 4D. + 2Db, where 0 is the angle between the Ca-H at the surface of the helix. The internal reorien• internuclear vector and the major axis of ellipsoid. tation of the side chain has been reported for For the PGA helix used here, a value of a is esti• esters of poly(glutamic acid) in non-aqueous solu• tion. 7, s, so mated to be 195 A from the degree of polymeri• Under the extreme narrowing conditions, the zation and that of b to be 7.5 A from a radius of the solvated helix. 27 The angle between the spin-lattice relaxation time of a protonated carbon, Ca-H vector and the axis of the helix is undergoing internal reorientation as well as iso• tropic overall reorientation, is given4 calculated to be 65° from the coordinates of the a-helix. 29 Using these parameters, we obtained l lz 2ro:rH2Tc1(A+ - Brint + _ C-r,n~ ) -rA =766 nsec, Tn=44.3 nsec, and r-0 =11.6 nsec at NT1 r '1nt+-rc1 '1n,+4,c1, 300 K. Equations 1, 3, and 7 with these cor• A=(3 cos2 ¢-1)2/4, B=3 sin2 ¢ cos2 ¢, relation times yield a value of T1 of 33 msec and C=3 sin4 ¢/4 (8) a value of NOE of 1.2. It is inferred from the combination of the values of T1 and NOE that the where -r,n.t is the correlation time of internal effective correlation time of the Ca-H vector fixed reorientation, -r 01 is the correlation time for re• to the ellipsoid is 23 nsec, which is greater than orientation of the axis of internal motion, and the correlation time of T1 minimum. ¢ is the angle between the C-H internuclear The correlation times calculated on the assump• vector and the axis of internal reorientation. tion of a rigid ellipsoid, determined by overall For the ,8-carbon, the internal rotation about motion of the molecule, are apparently longer than Ca-Cfi bond is allowed, and ¢ is a tetrahedral the measured correlation time. This suggests that angle. If the correlation time of the axis is the tumbling motion of the Ca-H vector is not assumed to be equal to the effective correlation only overall tumbling motion but also involves time for Ca, -r1n, for Ci3 can be calculated from appreciable local segmental motion of the back• eq 8. The correlation time for internal reorien• bone. This is clear from the fact that at pH 4.9, tation for the r-carbon was calculated by assuming PGA is a partial helix as mentioned previously. that r-01 for Cr is the effective correlation time for Using the effective correlation time of 2.8 nsec Ci3 which is estimated from eq 1 under extreme estimated from T1 and NOE, we can calculate the narrowing conditions. The results are summarized values of NOE and T2, which are found to be in in Table II at pH 5.1 and 7.5. It is found that the reasonable agreement with measured values as effective correlation time progressively decreases shown in Table I. in the order of Ca, Ci3, and Cr- The correlation When going from helix to coil with increasing time of internal reorientation also shows a similar pH, T1 and NOE increase, and the line width trend. It is seen that the internal motion of Cr decreases. All these observations are consistent carbon is about three times faster than that of Ci3 with a decrease in -rem as a result of the onset of the carbon. These results suggest that the end of the fast segmental motion of the polymer backbone. Table II. Effective correlation time (reff) and correlation time of internal reorientation

Ci3 and Cr Carbons (r1n,) at 300 K In all pH regions studied, as one goes away from the a-carbon of the backbone to the r-carbon Ca Ci3 Cr of the side chain, there is a progressive increase in pH• 'reff, l'eff 'Z"int T eff 'Z"int ns ns ns NT1 values and also in NOE, as shown in Figures

4 and 5. This increase in NT1 and NOE is due to 5 .1 0.96 0.53 2.7 0.26 0.94 the decrease in the correlation time of motions 7.5 0.65 0.35 1.5 0.16 0.55 in the sequence of Ca, Ci3, and Cr. These results • Direct meter reading in D 2O solution.

304 Polymer J., Vol. 11, No. 4, 1979 13C Magnetic Relaxation of Poly(Glu) side chain undergoes more rapid motions than others even in the helix state. This added degree of motional freedom is also convinced from InUn+l),n2 } the fact of the larger NOE observed for Cr carbon (9) InUn+l)rn2 as compared with the C., carbon. It is also seen that the internal motion of the Cfi and Cr carbons where r is the correlation time of C' carbon, are about two times slower at pH 5.1 than at pH In and In are the spin quantum numbers of 7.5, suggesting that the internal motions of the side• deuterium and proton, respectively, and other chain carbons in the helix state are more hindered symbols have been mentioned previously. As• than they are in the coil state. suming that r of C' carbon is identical to the correlation time of Ca carbon, and using the C' Carbon measured T1 value in H20 and D 20, the resulting distance is 1.98 A. This value is in good agree• Figure 4 indicates that the T1 value of the peptide C' carbon is an order of magnitude ment with value of 2.04 A calculated from bond greater than that of Ca carbon. This is due to distance and angle31 in spite of rough assumption. the fact that there is no proton directly bonded the C' carbon. The same situation is found for CONCLUSION Ca carbon. The 13C chemical shifts and relaxation times provided a reasonably detailed picture Table ill. Comparison of T1 value of C' carbon of the conformation in H20 and D20 in coil state at 300 K and motion of PGA in aqueous solution. Ca and C' resonances move upfield in conjunction with the helix-to-coil transition, while 0.88 1.28 Cfi, Cr, and Ca resonances move downfield in

• Direct meter reading in D 20 solution. conjunction with the ionization of the carboxyl group. These results are in good agreement with We measured the spin-lattice relaxation time those of Lyerla, et al. 19 The spin-lattice relaxa• of C' carbon in both D20 and H20 at neutral pH tion times and NOEs of protonated carbons in• to identify neighboring magnetic nuclei which crease when pH increases from 4.9. The T1 greatly contribute to the relaxation of C' carbon. values of Ca carbon indicate that even in the It was found that C' carbon relaxes more rapidly helix region at pH 4.9, PGA undergoes local seg• in H20 than in D20. Results are shown in Table mental motions. As moving away from the backbone, III. The measured T1 value is longer in D 20 the correlation times of the side-chain than in H20 by 45 %, It is reasonable to assume carbons progressively decrease, showing the pre• that the main contribution to the T1 value of C' sence of internal reorientation. It is found that carbon is the dipole-dipole interaction with a the spin-lattice relaxation of C' carbon is greatly directly bonded nitrogenc1 4N), a-proton which is affected by the dipole-dipole interaction with the two bonds removed, and an amide proton, also amide proton which is two bonds removed. two bonds removed. The amide proton is readily Acknowledgment. We are indebted to Dr. S. exchanged with deuteron in a D 20 solution. Mori of Ajinomoto Co. for kindly providing Therefore, the longer relaxation time in D20 is due poly(r-methyl o-glutamate). One of us (T.H.) is to the exchange of a proton for deuteron of the greatly acknowledged to the Japan Society for amide group. Because the gyromagnetic ratio of the Promotion of Science for the Research Fellow• deuteron (rn) is smaller than that of proton (rn), ship. This work was supported by the Grant-in• the 13C-2D dipole-dipole relaxation is less effective Aid for Scientific Research. compared to the 13C-1H dipole-dipole relaxation. In order to confirm this consideration we calculat• REFERENCES ed the distance between C' carbon and the amide proton. Under the extreme narrowing con• 1. G.D. Fasman, Ed., "Ploy-a-amino acids," ditions, the distance r will be given by Marcel Dekker, Inc., New York, N.Y., 1967.

Polymer J., Vol. 11, No. 4, 1979 305 T. HIRAOKI and K. HIKICHI

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