Carbon 13 Nuclear Magnetic Resonance of Pentapeptides of Glycine Containing Central Residues of Aliphatic Amino Acids*

SUMMARY Pentapeptides containing either glycine, L-alanine, L-valine, L-leucine, or L-isoleucine as central residue in the pattern glycylglycyl-X-glycylglycine have been studied by proton-decoupled natural abundance W Fourier transform nuclear magnetic resonance spectroscopy. Resonances were assigned by comparison with the free amino acids and by detailed observation of changes in chemical shifts with pH over the range 1.32 to 10.43. The effect of varying the central residue on the chemical shifts of the flanking glycine residues was established. Small effects ascribable to in-cipient aggregation were noted, both in terms of chemical shifts and relaxation properties. Each resonance was categorized according to the degree of sensitivity to the state of protonation of the terminal groups, and the pK values at 26” for those groups were determined from the pH dependence of the most sensitive resonances. The sensitive terminal resonances undergo broadening which is maximum near the PK. Spin-lattice relaxation times, T1, of protonated carbon nuclei were measured at two or more pH values in all cases except for the glycine pentapeptide. For

Small effects ascribable to incipient aggregation were noted, both in terms of chemical shifts and relaxation properties.
Each resonance was categorized according to the degree of sensitivity to the state of protonation of the terminal groups, and the pK values at 26" for those groups were determined from the pH dependence of the most sensitive resonances.
The sensitive terminal resonances undergo broadening which is maximum near the PK. Spin-lattice relaxation times, T1, of protonated carbon nuclei were measured at two or more pH values in all cases except for the glycine pentapeptide.
For each peptide C" resonances showed gradients in increasing T1 to each side from the central residue, indicative of contributions from segmental motion and internal rotation along the backbone in addition to the over-all rotational motion of the molecule.
Internal rotational modes are expressed as well in the aliphatic side chains, especially by the most peripheral methyl groups for which spinning around the attaching bond is clearly prominent.
Comparison of the anionic and cationic forms of the alanine, valine, and leucine peptides indicates that the rate of internal rotation of C" of the NHS-terminal glycine residues increases with deprotonation of the ammonium group.
The 13C nucleus acts in NMR experiments as a versatile and sensitive, nonperturbing probe of its physical chemical environment (2). Its chemical shift and relaxation properties offer a broad range of forms of chemical interpretation (3). NMR studies of a variety of compounds have illustrated that the W nucleus reflects not only the electronic features of its primary covalent linkage (4-6), but also the secondary influences of steric (7), electrostatic (8,9), and solvent (10) perturbations.
13C NMR studies of proteins (2, 11-14) with emphasis on 13C enrichment (15-23) can provide, in principle, a wealth of information concerning the structural and functional relationships that describe an organized polypeptide system. In practice the interpretation of 13C NMR of proteins is hampered by a lack of fundamental information about small peptides to serve as model compounds.
Presumably, the chemical shifts of the individual carbons which comprise amino acid residues in proteins can be described by a set of empirical terms that separately evaluate the effects of covalent linkage within the residue itself (9), as well as the effects of incorporating the residue into the primary polypeptide structure (24, 25), and the steric, electrostatic, hydrogen bonding, and solvent effects (18, 26, 27) arising from constraints imposed by the various levels of organization of the protein structure.
Although determining the resonance positions for the individual amino acids in the various protonated forms (9, 24, 28) represents a necessary first step in describing the expected range and classification of 13C chemical shifts, such assignments describe only qualitatively the resonance profiles for native and denatured proteins (11)(12)(13)(14).
More appropriate models are offered by short peptides (24,25,28) in which the effects of incorporation into peptide linkage can be emphasized and other effects such as end group ionization, vicinal electrostatic and steric interactions, peptide conformation, and unusual solvent conditions can be isolated or minimized.
The resulting assignments will serve as a reference for evaluating perturbations significant to protein structure. Previous work in this laboratory on tri-and pentapeptides established that terminal ionization effects are negligible for the central residue of the pentapeptide representing the NHS-terminal sequence of sperm whale myoglobin (24). The chemical shifts in proton decoupled spectra could be related to those of the constituent amino acids with systematic adjustments for incorporation into peptide linkages.
Proton coupled spectra were taken in certain cases to confirm the assignments. Characteristic chemical shifts of appropriate carbon nuclei were found to conform to theoretical titration patterns.
The present work is an extension of that study to a series of pentapeptides in which each common protein amino acid except cysteine is prepared as the central residue flanked by 2 residues of glycine in the general form Gly-Gly-X-Gly-Gly (29). Measurements of chemical shifts have been made over a range of pH to encompass all ionizations.
In addition, spin-lattice relaxation times, T1, have been measured.
The present report deals with the aliphatic amino acids as central residues.
EXPERIMENTAL PROCEDURE Peptide Synthesis-The amino acids L-alanine, L-valine, L-isoleucine, and L-leucine (Pierce Chemical Company) were converted to the respective t-1308 derivatives according to the method of Schnabel (30) by treatment with t-butylazidoformate (Pierce). This procedure was also employed in the conversion of glycylglycine (Pierce) to the t-BOC form by reaction in dioxane-Hz0 (1: 1, v/v) at pH 9.5 for 9 hours. The product was recrystallized from hot ethyl acetate and melted at 131-132' (31). Pentaglycine was purchased from Cycle Chemical Corp. and was used without further purification.
The pentapeptides were synthesized by the Merrifield solid phase technique (32) as outlined by Stewart and Young (33). A three st,age procedure was employed whereby t-BOC-glycylglycine was initially coupled to the chloromethylated resin (Bio-Beads S-X2,200 to 400 mesh, Bio-Rad) by reaction with triethylamine in absolute ethanol for 30 hours. The specific t-BOC-L-amino acid was then coupled to the resin-bound diglycine (3.5 meq of peptide per 7 g of resin) with N, N'-dicyclohexylcarbodiimide (Pierce), and finally t-BOC-glycylglycine was added to complete the synthesis by carbodiimide activation.
The swelling solvent was methylene chloride and the t-BOC adducts were removed in 30 min by 50% trifluoroacetic acid-methylene chloride (v/v).
A mole ratio of t-BOC amino acid or t-BOC-glycylglycine to resin-bound peptide of 2.5 to 1 was employed with coupling times of 2 hours.
In the final stage t-BOC-glycylglycine was dissolved in methylene chloride-dimethylformamide, 4 : 1 by volume.
The finished resin-bound t-BOC-pentapeptides were deblocked and cleared from the resin by bubbling HBr into an anhydrous trifluoroacetic acid suspension of the peptide-resin for 60 to 90 min. The HBr was passed through a scrubbing solution containing 2 g of resorcinol in 100 ml of anhydrous trifluoroacetic acid. The liberated peptide was collected in 250 ml of anhydrous trifluoroacetic acid and dried by rotoevaporation. The resulting oil was washed three times with 100 ml mixtures of methanol and water, 1 :l by volume.
Finally, the peptide was dissolved in a minimum amount of water and extracted five times with equal volumes of absolute ether. The aqueous solution at pH near 2 was dried, and the peptide was stored in a desiccator at 4". Yields were approximately 80 %. Peptide aliquots were hydrolyzed by redistilled 6 N HCl in evacuated glass tubes at 110" for 24 to 72 hours. Analyses were obtained with a Beckman 121 amino acid analyzer.
The expected mole ratio of 4:l glycine to amino acid was obtained. The peptides were homogeneous on thin layer chromatography (Silica Gel S, J. T. Baker) developed with 2-butanone-acetic acid-water, 2 : 6 : 5 v/v. Sample Preparation-Each peptide, 0.5 to 1.0 g, was dissolved in a minimum amount of deionized, distilled water, approximately 1 The abbreviations used are: &BOC, tert-butyloxycarbonyl; T1, spin-lattice relaxation time; NOE, nuclear Overhauser effect.
2.5 ml, containing 1 to 2 drops of dioxane as an internal NMR standard.
Measurements of pH were made before and after each NMR experiment on a Radiometer pHM4c meter equipped with a GK2302C combination electrode. All pH measurements were obtained at 26", and pH adjustments were made with either 6 N HCl or 5 N NaOH.
Standard buffers at pH 4,7, and 9 were used to calibrate the pH meter.
laC NM&--Most experiments were performed on a "homebuilt," pulsed Fourier transform spectrometer equipped with a Varian 14.1 kG electromagnet operating at 15.08 MHz. A detailed description of this apparatus has been published elsewhere (11).2 Magnetic homogeneity and frequency settings were adjusted using neat ethylene glycol.
All chemical shifts are reported upfield of CS2 with the aqueous dioxane resonance located at 126.3 ppm and external neat ethylene glycol at 129.7 ppm. Chemical shifts for titration experiments were measured directly from locations in computer memory.
In most cases the "observing window" was 250 ppm giving a spectral resolution of 0.1 ppm. In some cases the Varian XL-loo-15 instrument was used as specified in the text. Partially relaxed Fourier transform spectra were obtained on the 15.08 MHz instrument by the Inversion-Recovery Method utilizing a 180"~t-90" pulse sequence (34). Temperature variation within a given relaxation set was zkl".
All spectra were obtained between 24.5 and 33.5" as indicated in the text. Unless otherwise stated the observing window was 125 ppm and recycle times were at least three times the longest measured T1 values.
Calculations-The titration chemical shifts were fit to the Henderson-Hasselbalch equation using a computer program designed to minimize error by the Powell (35) method of conjugate directions.
Each point in the fit was weighted according to the sum of the errors in spectral resolution and in pH, estimated to be 0.1 ppm and 0.02 pH units, respectively.
The pK values reported by this method are accurate to the first decimal place.
Values for T1 were obtained from computer analyses of the relaxed spectra as described elsewhere (11). Computed standard deviations fall in the range of 2.5 to 5%.

RESULTS
General Characteristics of Spectra-Illustrative spectra of the five pentapeptides containing an aliphatic central residue are presented in Fig. 1. The experimental conditions are described in the legend and are representative of those used for other experiments except as mentioned specifically below.
With the exception of the spectrum in Fig. 1D for the leucine peptide which was obtained at pH 6.98, all these spectra were taken in the pH range 1.32 to 2.10. In each spectrum the dioxane resonance appears at 126.3 ppm upfield of CSZ. Downfield of dioxane the spectra show only the group of resonances representing the four peptide carbonyls and the terminal carboxyl carbon nucleus.
In the upfield region fall all Ca resonances and those of the aliphatic side chains. Within each chemical class of protonated carbon nucleus the resonance area defined by signal height and line width generally reflects the numbers of resonating carbons.
Note should be taken of certain sources of variability of attenuation of resonance intensity in the spectra.
For example, 2 The development and construction of the l3C Fourier transform nuclear magnetic resonance equipment was supported in part by grants to Adam Allerhand -from the National Science Foundation (GP-17966) and the Petroleum Research Fund (4559-AC5). ~ Comparison of chemical shifts in amino acids and peptides Chemical shifts of aliphatic amino acids in free dipolar form are compared with those incorporated as the central residue of a dipolar pentapeptide of the form Gly-Gly-X-Gly-Gly. Carbon types listed use the IUPAC-IUB nomenclature.
All chemical shifts are expressed as parts per million upfield of C&. relaxation times for carbonyl carbon nuclei in the pentapeptides are usually so long that it has been impractical as a rule to recycle slowly enough to avoid signal attenuation.
Variations in the efficiency of 'H-decoupling may make a minor contribution to relative attenuation.
Limitations in computer memory also restrict the comparison of intensities involving the narrow, nonprotonated carbon resonances.
There is no evidence of significant differential broadening of the carbonyl or Car resonances by the bonded 14N (36). Regardless of pH, no slow exchange effects are seen either in terms of sudden changes in signal to noise or with respect to the appearance of multiple resonances for the same carbon nucleus. As will be discussed further below, certain resonances that are sensitive to titration of the terminal residues undergo a broadening in the pH range near the pK of the titrating group.
Chemical shift overlap is sometimes observed for the carbonyl nuclei of the glycine residues adjacent to the central residue, and is the rule for C" of those residues so that a prominent landmark is recognizable in that region of each spectrum.
Spectral assignments have been made on the basis of comparison with the free amino acids and by following stepwise the effects of variations in pH, and also are supported by measurements of spin-lattice relaxation times.
In some cases resolution has been expanded to define overlapping peaks. Complete lH-decoupling was maintained throughout this work since assignments could be made without resorting to partial decoupling techniques.
For convenience in illustrating the later discussion the assignments in Fig. 1 are identified by numbering the residues 1 to 5, starting at the NHS-terminal.
Proceeding upfield in the carbonyl region the 5 residues fall in the case of the glycine peptide in the order of residue 5, followed by residues 2, 3, and 4 overlapping in the dominant resonance, and lastly residue 1. In the cases of the alanine, valine, and isoleucine peptides the order is residues 3, 5,4, 2, and 1. In the case of the leucine peptide at pH 6.98 the order is 5,3,2,4,1.
In the Ca range, excepting again the leucine peptide, the order is 3, 2, 4, 5, and 1, with 2 and 4 overlapped as mentioned above. With the leucine peptide the Cm of residue 5 is shifted enough at pH 6.98 that the order is 3, 5, 2, 4, and 1. With the glycine peptide Cal of residues 2, 3, and 4 all overlap, although the upfield shoulder of this band is clearly resolved into a single carbon resonance at 25.19 MHz.
The side chain carbon nuclei fall progressively upfield according to their distance from Ca.
Chemical Shifts of Central Residues-For convenience in bringing out the systematic patterns the results for the varying central residue in the pentapeptides are presented first, followed by those of the flanking and terminal glycine residues.
Table I compares the chemical shifts for the free amino acids involved, obtained for the dipolar form in each case, with those for the central residues of the pentapeptides, the latter also in the dipolar form in each case. Columns are given for the respective chemical shifts Spcptide and 6rree and for their difference, (&ptide-G&.
The value of this difference reflects the effect of incorporating the amino acid into the peptide form. The effect varies for the different residues involved.
The relationships of the chemical shifts within the residues are consistent with known substituent effects (2,4, 5, 9), excepting the recognized nonequivalence of the methyl groups in valine, leucine, and isoleucine (9, 24). Presumably the effects of near neighbors in the peptide sequence on the chemical shifts of the central residue are minimized by the choice of glycine for the flanking residues.
Ti&ation Eflects-Chemical shifts of the flanking and terminal glycine residues are presented conveniently in terms of their pH dependence.
The variation of chemical shift of these glycine residue carbon nuclei with pH is summarized in Table II. The values at basic pH and at acid pH, 8s and SA, respectively, are shown, as well as their difference.
Because of the wide separation of the titrating groups the deprotonation at one end of the molecule does not influence the chemical shifts at the other end.
In keeping with earlier results (24, 25) the largest values of (8~ -6,) with increasing pH are -8.4 and -3.1 ppm for the Co and C", respectively, of the glycine residue 1, and -3.2 and 6107 -2.15 ppm for the C" and C", respectively, of glycine residue 5. The titration effects are not limited to the terminal residues, particularly for the cases of the C" of residues 2 and 4. Notably the C" of glycine residue 4 exhibits an upfield shift with deprotonation of the terminal carboxyl group. By expanding the resolution evidence of two peaks for the nearly identical resonances of Ca in residues 2 and 4 may be obtained in the midrange of pH, but the distinction is less than 0.05 ppm.
The fit of the chemical shifts to theoretical titration curves is illustrated in Fig. 2 for the glycine residues. The curve for the central carbonyl has been included in each case to emphasize the magnitudes of the shift range and the spectral overlap encountered in the individual titrations.
Clearly the ionic strength was high enough in all cases that variations in it were without observable effect. The method of sample preparation was such that titrations were commenced from the acid rather than the neutral region. However, enough difficulty was encountered with solubility of most of these peptide solutions that the pH values were often approached from different sides, with attendant small irregularities in the trend of electrolyte concentration along any given titration curve. The insensitivity to variation in ionic strength of the 13C NMR spectra of such compounds under comparable conditions has been reported (24).
The pK values corresponding to the curves of Fig. 2 are collected in Table III.
The average pK values computed by weighting according to the magnitude of (8s -6*) are listed with the designation of the peptide, followed in the next column by the temperature of measurement.
by the various carbon nuclei are clearly coherent. As described previously (24), in the absence of overlapping titration ranges the pK values determined are those for the temperature of the pH measurements, here 26.0". The observed values are in the expected range (24, 37). Several of the pK values in the table are designated with superscripts to show that although the results fit the respective pK value adequately the (6~ -6,) value was too small for an accurate computation to be made. The magnitudes of these shifts were approximately 0.1 to 0.2 ppm. Those cases are indicated in which no evidence of pH dependence n-as seen. Note is also made of marginal effects of pH variation indicating very slight chemical shift changes eit,her in a titration range or in the region of the p1.
As the resonances of the carbon nuclei of the terminal residues shift downfield with increasing pH broadening is observed, max-imally near the pH corresponding to the pK. For the Crr resonances this broadening is less clearly made out than for C" because the titrating resonances near their pK are overlapped by the intense C" resonance of the residues 2 and 4. However, the reduction of the peak maximum in this range may be observed even in the face of considerable overlap (see Fig. 1E). The broadening is clearly visible for C" nuclei, especially that of residue 1. Even at one pH unit below the pK the broadening can be detected and there is usually a reduction in peak height of at least 20 to 30%.
The effects of incorporating the glycine residues into the terminal positions of the pentapeptide can be described in the same terms as were used above for the central residues (Table I).
Taking the chemical shift values for dipolar glycine from Table  I, and drawing on the values for the dipolar forms of the pentapeptides, it is seen that the incorporation produces a shift of +4.5 and -4.1 ppm, respectively, for C" nuclei of the residues 1 and 5. As already pointed out, the incorporation into the central residue produces a shift of only $0.4.
The neighboring effect of the varying central residue in the pentapeptide series causes a variation in the corresponding shift differences for the respective flanking glycine residues 2 and 4, falling in the range of +0.5 to 1.5 ppm. The magnitudes of these shift differences reflect the combined effects of the variable central residue and the other neighbors that are the two different terminal glycine residues.
The small but notable differences in resonance position for the carbonyl of glycine residue 2 in these peptides at acid pH and of glycine residue 4 in alkali illustrate the magnitude of the "nearest-neighbor" effect of the central residue. The analogous effects on C" nuclei of glycine residues are smaller than those for C": $0.85, -1.75, and -0.9 for the residues in positions 1, 5, and 2+4, respectively.
The effect of variation in the nature of the central residue on CP of residues 2 and 4 is small.
The resonance positions of nuclei of glycine residues reported here are consistent with effects predicted from studies of tripeptides (9,24, 25) and from substituent parameters (4, 5, 8).
Xpin-lattice Relaxation Times, Tr--Tl values were measured at more than one pH for each pentapeptide except pentaglycine. The conditions of measurement did not permit accurate determinations of the T1 values of the carbonyl carbon nuclei.
The long T1 values encountered, of the order of 3000 to 7000 ms, require long periods of data collection for comparable accuracy. There are also experimental limitations on the accuracy of measurement for long T1 times (3, 38). Moreover, the mechanisms' of relaxation of the nonprotonated carbon nuclei are not clear (3, 39, 40). For these reasons the results presented in Fig. 3 are limited to the proton-bearing carbons, set out in a schematized form for ready identification of the individual backbone and side chain carbon nuclei.
Following the style of a preliminary report (29), the relaxation times are written in at the location of the corresponding carbons in the structural scheme, and are expressed as NT1 values in which N is the number of directly bonded hydrogen atoms.
This practice allows for the approximately additive effect of each hydrogen in contributing to the dominant %YH dipole-dipole relaxation mechanism (41). For ready identification of the charged state of the peptide each diagram shows the terminal amino and carboxyl groups in the appropriate ionized forms. Conditions of temperature and pH are given in Table IV.
The dominant dipole-dipole relaxation mechanism determines a dependence of NT1 on the effective rotational correlation time for the protonated nucleus in question (41,42 for protonatrd carbon m&i. The values in Fig. 3 are arrsnged according to carboll assignments based OIL titrat'ionnl behavior and substituent additivity rules (4, 5, 8) and geiwally follow quite prrdictably the corrclation of longer 5'1'1 valw v it11 illcreased distance f'~~;~ll the central' Ca. Since the Ca Jlwlei of glycille residues 2 and 4 arc usually poorly resolved, their average value has been indicated in most cases. These two nuclei nlqx=ar to relax similarly, as judgtd by The same 1:attcrn applies to the lxogression of carbon nwlci out the side chains.
The Kl'l values for the majority of nuclei in a given pcptidc in this aliphatic series at neutral $1 are lower than those for either pH extreme.
This observation suggests that aggregation of these pcptides tends to owur near neutrality.
This bchn~ior is consistent with the slight but unmistakable 111-I trends JIHWtioned preriously for the rrsonances of the central residues, as well as with the titrational lxolwrtics of the terminal groups. It' is also consistent with the observed decrease in solubility of these l:eJltapeptides when predominantly in the neutral f'ol m. Interconversion among the peptide forms is fast, as judged by the resonance intensities and line widths.
The largest values of NT1 in Fig. 3 arc observed for the methyl groups.
Here the spinning motion about their axis of attachment is espcctcd to make a major or dominant cont,ribution. Comparison of the NT1 for methyl groups in Fig. 3 Tyith those for the corresponding Ca show in several cases a ratio approaching the theoretical limit of 9: 1 deduced for a freely spinnilrg methyl group attached to a large, rigid matris tumbling isotropically with an appreciably longer correlation time (42). It is significant that the correlation is most obvious in thaw cases where aggregation is suspected, eslwially with the lrwine and isoleucine peptides.  Table  IV. atoms.
The NT1 values are written in the location of the cor- the responsive terminal glycine residues appear to broaden, going through a maximum line width near the pK and having minimal line width at least 2 pH units above or below the respective pK value.
Integrated areas of resonances at the pH extremes correspond quite well with the number of nuclei involved.
The line width of the NHz-terminal Ca resonance appears to be significantly increased at pH 6.70 for the isoleucine pentapeptide compared with ~1% 4 or 10. The line width was approximately 27 Hz at pH 6.70 compared with 7 Hz at low and high pH.
Separate measurements of integrated areas in the presence and absence of complete 'H decoupling at 25.19 MHz at pH 6.70 showed that the apparent reduction of NT1 for the terminal C" (Fig. 3) was accompanied by a reduction in the nuclear Overhauser effect (NOE) from 2.9 to 2.0, whereas all other protonated carbon resonances exhibited maximal NOE values3 Integrated areas for NHz-terminal Ca resonances in the other peptides at neutral pH were much less decreased.

DISCUSSION
The choice of the pentapeptide model with the residue under temperature in the range from 26-33".
The values for the other study in the central position flanked by pairs of glycine residues nuclei increase over this temperature interval.
was made with the purpose of having the simpiest possible refer-The three sets of results for the alanine peptide in Fig. 3 at ence position for the central residue (44)(45)(46). This peptide pH 1.30, 6.87, and 9.80 offer the most clear-cut example of the design should yield a consistent comparison of chemical shifts effects of full ionization of the terminal groups.
Deprotonation of the a-amino group appears to increase NT1 for Ca of glycine of the various components of the different amino acid residues respond to effective correlation times, doff, of about 1 to 3 x introduced in the central position.
Such results are set out in lo-lo s. This range is nearly the same as that for denatured Tables I and III. The good correlation observed between the proteins (11, 12, 15, 50) and falls slightly below the value ob-Henderson-Hasselbalch equation and the titration profile of taincd for isotactic polystyrene of molecular weight greater than those resonances sensitive to the state of protonation of near lo4 (51). The rotational motion contributing to the relasation neighbors (Fig. 2, Tables II and III) indicates that there are no processes for protonated carbons in these macromolecules has strong interactions shown by these peptides that result in asym-been interpreted in terms of the dominance of segmental motion. metrical titration behavior (47). On the other hand, small The present trend of increasing NT1 of the backbone CP nuclei, effects were noted above involving very small changes in chem-especially the large values for the terminal Ca nuclei, probably ical shift of the central residues during titration.
The interpre-reflects significant contributions from internal rotation. Thus, tation that these marginal effects are caused by aggregation the motion of the pentapeptide backbone is presumably deappear to be supported by the T1 measurements (Fig. 3, Table  scribed best in terms of over-all reorientation, segmental motion IV).
The chemical shifts and relaxation behavior are pre-and internal rotation. The complexity of this motion prevents sumably sensitive here to a minor degree of aggregation, far too any detailed interpretation (42) in terms of specific correlation little to show systematic line broadening under the conditions times for distinct motional modes since all these values are of the experiments or to cause noticeable light scattering.
One likely to be similar in magnitude.
The effects of internal motion should be alert to the possibility that in the compact native are much less evident for the side chain nuclei of the central protein structure the corresponding effects on chemical shifts residues, with the exception of the methyl groups. resulting from bringing various residues into contact may be Peptide association tends to reduce the NT1 values of the appreciably greater.
Most of the other pentapeptides in this nonterminal carbon nuclei by virtually the same percentage series, to be reported separately, are less amphipathic and have (Fig. 3). The terminal C" nuclei are affected much less and the presented fewer problems with solubility; such effects have been side chain terminal methyl groups are the least responsive of all much less evident with them. the nuclei. Separate measurements of NT1 values of the leucine The observations on residues 2, 3, and 4 in the glycine penta-peptide at pH 9.88 and 33", compared with those at pH 9.73 peptide show that, within limits, the chemical shifts of residues and 25.8', illustrate that the backbone and side chain carbon 2 and 4 are only moderately sensitive to terminal ionization nuclei, except for the methyl groups, show virtually an identical effects, a point stressed more generally by the results of Christ1 percentage increase in NT1 with increased temperature (by and Roberts (25). As a result the pattern of variation of the approximately a factor of 2). Notably the methyl groups excentral residue from one peptide to another can be used to follow hibit identical values of NT1 regardless of temperature. These the effects of the neighboring substitution on the glycine residues results suggest that all the carbon nuclei except those of methyl 2 and 4. Table II shows that in terms of chemical shifts these groups are influenced significantly by the over-all rotation of effects are small for Ca in those residues but are considerable for the molecule. The contribution of 7R to 7eff is marginally less C", especially in residue 2. Sot surprisingly all these aliphatic at the terminal OL carbons and is virtually absent at the methyl substituents have similar effects. To anticipate, somewhat groups of leucine. Presumably the motion of the peripheral larger effects have been observed in this w-ay with other penta-methyl groups is dominated by rotation about their axes of peptides, and will be reported sel>arately.4 attachment to the side chain. The characteristic pK values for these pentapeptides exhibit a The trend of increased NT1 values of the methyl groups with greater variability for the terminal carboxyl group compared increased distance from the central C" may be characterized by with the terminal amino group. This greater variability is not the ratio of the NT1 value for the methyl group to the NT1 value the result of ionic strength differences and cannot be explained for the central residue Ca. These ratios are approximately 4, 5, strictly in terms of experimental temperature variations (see 9, 11 for the most peripheral methyl groups of alanine, valine, Table III) since the measured thermal coefficients over this leucine, and isoleucine, respectively. The maximum ratio for a temperature range are expected to be small (48). The carbosyl freely rotating methyl group attached directly to a rigid backterminal pK values follow a n-eak trend toward elevated values bone undergoing isotropic reorientation is 9 (42). Clearly the as the size of the central residue increases.
backbone for any of these pentapeptides does not conform in The spin-lattice relaxation times, T1, show meaningful trends its motional properties to the criterion of "isotropic rigidity" as within each peptide.
Since the %JH dipolar mechanism is would that for a cyclic peptide (27, 36, 52-56), so that a direct dominant in most cases and the extreme narrowing limit applies interpretation of these NT1 values in terms of correlation times here, the values of NT1 are related to a first.approximation to for internal motion is not strictly correct (42). However, thethe inverse of an over-all rotational correlation time, 7n (42). oret.ical calculations for dipeptide units containing glycine and The simplest relation is reserved for the case of isotropic tum-alanine (57,58), valine (59), leucine (60), and isoleucine (57) bling (41,42). In applications to small molecules (49) and indicate that /3 and y methyl groups interact with the peptide especially proteins (50) it has been helpful to draw attention backbone, whereas the 6 nuclei do not. These interactions to the multiplicity of motional components in the behavior of have the consequence of restricting the available rotational a given nucleus by substituting the effective correlation time, angles of the backbone cp and # angles and also serve to restrict Teff, for rR (2, 11, 12, 42, 49). Identity of NT1 values, there-the configurations of the associated methyl groups. Our trends fore, does not prescribe identical combinations of components in NT1 ratios are consistent with these predictions and compare of motional behavior.
However, within limits useful compari-satisfactorily with results from x-ray analysis of lgsozyme, myosons can be made.
globin, and oc-chymotrypsin (61)(62)(63). This evidence for the The NT1 values for the Ca nuclei of the central residues cor-dominance of the rotational motion of the most peripheral Comparison of the NT1 values of the respective pentapeptides in the acid and alkaline forms (Fig. 3) illustrates that the values for the NHz-terminal Ca nuclei are much increased at high pH compared with those for all other protonated carbon nuclei. Since the NOE is maximum for these carbons, the YYH dipolar mechanism is dominant.
These results show that the NHtterminal Ca undergoes a change in rotational motion with deprotonation of the ammonium group. The simplest interpretation is that the rate of internal rotation of the KHz-terminal Ca increases with deprotonation of the ammonium group. This response is consistent with the expected reduction in solvation of the neutral amino group compared with the positively charged ammonium function (64). Similar features have been observed in studies of the molecular dynamics of glycylglycine (65). The interpretation for the Ca of the carboxyl terminal is less clearcut, since the indicated increases in NT1 at high pH approach experimental error. The results listed in Table II illustrate that the C" of glycine residue 4 shifts upfield with deprotonation of the terminal car-boxy1 group.
In contrast all other resonances sensitive to terminal ionization shift downfield with deprotonation. Similar upfield shifts have been observed for the carbonyl of the amino acid units directly attached to the carboxyl terminal residue in di-and tripeptides (24, 25). Such results have been interpreted to reflect possible contributions from protonated forms of the peptide bond carbonyl at low pH. The suggested mechanisms include full protonation of the peptide bond (66)(67)(68), hydrogen bonding with solvent (69) or intramolecular hydrogen bonding between the peptide carbonyl and the terminal protonated carboxyl group (69). Alternatively, these upfield shifts may be evidence for the formation of a &gauche conformation at neutral pH stabilized by an electrostatic interaction between the terminal carboxylate and ammonium groups (70). Our results are not compatible with most of these features.
Proton charging of peptide bonds is unlikely under the present conditions (66). Since the upfield titration effect is observed at only one of four peptide bonds, nonspecific hydrogen bonding with solvent appears unlikely.
The upfield shift is not evidence for a folded conformation stabilized by electrostatic forces since the responsive carbonyl resonance should shift downfield with deprotonation of the NH2 terminus.
Our results tend to favor attributing this titration effect either to charge or steric effects associated with the state of protonation of the terminal carboxyl group or to hydrogen bonding at the peptide carbonyl directed by the terminal protonated carboxyl group.
For the single case of the isoleucine pentapeptide at pH 6.70, the measurements of T1, line width, and NOE most likely point to an additional relaxation mechanism, competing with the 13C-1H dipolar effect and coming into play near the pK of the terminal ammonium group.3 Spectral broadening has been observed in this laboratory for a 'V-enriched glycine adduct attached to sperm whale myoglobin (19) and for the C@ of aspartic acid in pentapeptide form.4 The trends in line width discussed specifically for the isoleucine pentapeptide and observed generally for all the pentapeptides reported here are similar to trends which occur in the line width for the o(-CH protons of polyamino acids during helix-to-coil transitions (71). Transition exchange rates calculated from the LU-CH proton line widths are much slower than those based on other techniques (72). Proton exchange rates calculated from the o(-CH proton line widths of model peptide systems in which the peptide bond is protonated by strongly acidic organic solvents (66,67) are similar to the values reported from the NMR studies of helix-to-coil t.ransitions induced by increased amounts of strong organic acids (68,71). A rate constant determined (71,73) from the corrected line width of the Ca of glycine residue 1 of the isoleucine pentapeptide falls in the range associated with proton exchange rates (66,67,74,75) in the peptide systems described above. The characterization of this effect in other pentapeptides will require the study of isolated resonances over the entire pH range at different resonance frequencies.