Effects of Hydrogen Bonding and Temperature Upon the Near Ultraviolet Circular Dichroism and Absorption Spectra of Tyrosine and O-Methyl Tyrosine Derivatives*

SUMMARY To gain information about the properties of tyrosyl residues buried within proteins, the circular dichroism (CD) and absorption spectra of tyrosine derivatives have been investigated in nonpolar solvents. N-Stearyl-L-tyrosine n-hexyl ester dissolved in methylcyclohexane (O-O band at 283 nm) was used to measure the effects of hydrogen-bonding agents. Adding l- shift in the a in dipole The results N-dimethylacetamide between a hydroxy and a carbonyl of the peptide be

From the Laboratory of Nuclear Medicine and Radiation Biology, University of California, Los Angeles, California 90024

SUMMARY
To gain information about the properties of tyrosyl residues buried within proteins, the circular dichroism (CD) and absorption spectra of tyrosine derivatives have been investigated in nonpolar solvents. N-Stearyl-L-tyrosine n-hexyl ester dissolved in methylcyclohexane (O-O band at 283 nm) was used to measure the effects of hydrogen-bonding agents. Adding low concentrations of dioxane, N, N-dimethylacetamide, 1-butanol, or methanol causes a l-to 4-nm red shift in the absorption spectrum and a 10 to 25% increase in the dipole strength.
The results with N, N-dimethylacetamide suggest that a hydrogen bond between a tyrosyl hydroxy group and a carbonyl oxygen of the peptide backbone may be one mechanism for producing a large red shift in proteins. CD spectra were recorded after the hydroxy group of N-stearyl-L-tyrosine n-hexyl ester had been hydrogen bonded. Dioxane and N, N-dimethylacetamide cause the CD spectra to red shift and intensify to the same extent as do the absorption spectra. Evidently hydrogen bonding to these compounds does not alter the conformation of this tyrosine derivative. In contrast, hydrogen bonding of N-stearyl-L-tyrosine n-hexyl ester to butanol or methanol causes a 50% loss of rotatory strength, suggesting an altered conformation.
The dependence upon alcohol concentration is the same for both the CD and absorption alterations (halfmaximal effect at 30 mM alcohol). Evidence is presented * This investigation was supported by Contract AT(04-1) GEN-12 between t,he Atomic Energy Commission and the University of California. that a polymeric form of the alcohol may simultaneously hydrogen bond to both the hydroxy group and the amide oxygen atom of N-stearyl-L-tyrosine n-hexyl ester. At concentrations greater than 15 PM, N-stearyl-L-tyrosine n-hexyl ester is partially aggregated in methylcyclohexane, causing changes in the CD and absorption spectra. The aggregate is proposed to be mainly a dimer in which the hydroxy group of each tyrosine residue is hydrogen bonded to the amide oxygen of the other residue  Information  concerning  the circular  dichroism  spectra of  model compounds  is a prerequisite  to understanding  the near  ultraviolet  CD" bands of proteins.  Recently  the CD spectra  of several tyrosine derivatives  have been investigated  both experimentally  (l-4) and theoretically (5, 6). The theoretical studies clearly showed that the near ultraviolet tyrosyl CD bands are strongly conformation dependent (5, 6). Unfortunately, predicting the conformation from the observed CD bands is usually difficult because of the many conformations possible for nonrigid molecules.
In addition, the relative populations of these conformers may be influenced by solvent-solute interactions, such as hydrogen bonding, and by temperat'ure. This communication describes the CD spectra of several tyrosine derivatives which were synthesized so that the effects of temperature, solvents, and hydrogen bonding could be investigated in detail.
N-Stearyl-n-tyrosine n-hexyl ester is soluble in both nonpolar and polar solvents, but is prone to aggregate. N-Stearyl-0-methyl-n-tyrosine n-hexyl ester and N-acetyl-Omethyl-n-tyrosine ethyl ester do not aggregate as readily and were, therefore, more satisfactory for examining CD spectra at low temperatures.
The effects of hydrogen bonding upon the CD and absorption spectra were studied with the use of both N-stearyl-n-tyrosine n-hexyl ester and its corresponding Omethyl derivatives.

MATERIALS AND METHODS
instrumentation-CD spectra were recorded with an instrument that has been described in detail elsewhere (7,8). Reliablc, low noise CD records were obtained by averaging four to 32 scans at 0.3 nm per see with a Fabri-Tek model 1072 signal averager (8,9). The CD intensity was calibrated with an aqueous solution of d-10.camphorsulfonic acid (AE = 2.2 M-l cm-l at 290 nm (lo), where A E is the molar extinction coefficient for left circularly polarized light minus that for right circularly polarized light).
The techniques for low temperature CD measurements have been described previously (2). In calculating the AE values at, each temperature, the concentration increase upon cooling was taken into account by using the volumecontraction data for EPA (11) and for methylcyclohexane (12). Unless otherwise indicated, the concentration dependence of these CD spectra was in accordance with Beer's law over at least a 5-fold concentration range. These findings imply that aggregation is not responsible for the changes in CD spectra observed for X-Ac-0-Me-n-Tyr ethyl ester and N-AC-L-Tyr ethyl ester upon cooling.
Most absorption spectra were recorded on a Cary model 15 spectrophotometer.
In some cases, however, a direct comparison was made between the wave length positions and shapes of the CD and absorption bands by recording absorption spectra on the CD spectrophotometer.
This procedure was necessary because the spectral resolution of sharp bands is somewhat poorer on the CD instrument.
To record these absorption spectra, a portion of the photomultiplier dynode voltage was fed through a log amplifier ) and then to the input of the signal averager.
The absorption spectrum was obtained by recording the log of the sample voltage in the memory units of the signal averager and then subtracting the log All other solvents were spectroquality.
The methylcyclohexane did not have any near ultraviolet absorbing impurities (5.cm path length). N-Ac-0-Me-n-Tyr ethyl ester was synthesized by the acetylation of 0-methyl-L-tyrosine ethyl ester hydrochloride with 3-fold excess of acetic anhydride in 1 M sodium bicarbonate. The precipitate was collected and crystallized from ethyl acetate (m.p. 93").
CdbNO~ Calculated: C 74.57,H 10.73,N 2.63 Found : C 74.45,H 10.55,N 2.48 The E value in methylcyclohexane was taken to be the same as that of N-Ac-0-Me-n-Tyr ethyl ester. N-Stearyl-n-phenylalanine n-hexyl ester was prepared from stearic acid and n-phenylalanine n-hexyl ester hydrochloride (m.p. 127O). The product was semicrystalline.

Comparison
of p-Cresol and p-Methylanisole Spectra-As a preliminary step, we compare the near ultraviolet chromophores of 0-methyl-r,-tyrosine and of L-tyrosine. The most appropriate model compounds for these chromophores are p-methylanisole and p-cresol, respectively.
The vibronic structure of their absorption bands can be clearly seen by using perfluorinated hexane as the solvent.
As shown in Fig. 1, the wave length positions and spacings of the vibronic bands are essentially identical in p-methylanisole and in p-cresol when dissolved in this relatively inert solvent.
These high resolution solution spectra reveal the same major vibronic bands as have been reported in the Tyrosyl CD ancl Absorption Spectra Vol. 247, No. 2 /11111,,,,,,,,,,,,,,/,,,,,,,,,,,   This solvent, which has been used for previous studies of tyrosine and tryptophan derivatives (2), was chosen because it remains liquid down to very low temperatures. As shown in Fig. 2, at 297 K N-Ac-O-1Me-L-Tyr ethyl ester has weak, positive CD below 273 nm and weak negative CD at longer wave lengths.
Upon cooling to 215 K, the negative portion of the CD spectrum is enhanced.
At 140 K the CD spectrum is further intensified, and vibronic bands are well resolved at 283 and 277 nm (Fig. 2). Cooling to 140 K causes a IO-fold increase in the negative rotatory strength over that observed at 297 K. As expected, the vibronic bands ( This band sharpening occurs without any measurable change in the dipole strength of the near ultraviolet absorption band (less than 3yc intensification).
Since EPA is a mixed solvent containing both an alcoholic and ether component, the CD spectrum of N-Ac-0-Me-L-Tyr ethyl ester was also examined in dioxane and methanol at room temperature.
The CD spectrum of this compound is positive in dioxane and negative in methanol ( Fig. 2). hdditional evidence relating to the variability of CD intensity was obtained from N-Ac-0-Me-L-Tyr ethyl ester dissolved in methylcyclohexane.
In this solvent, N-Ac-0-Me-L-Tyr ethyl ester possesses an intense, positive CD spectrum with pronounced vibronic structure even at 297 K (Fig. 3). The sharpness of these bands is further enhanced by cooling to 225 K, and the rotatory strength is increased an additional 359. (Fig. 3). The sharpness and intensity of these bands at 225 K provide an exceptional opportunity to examine the correspondence between CD and absorption bands.
To make an accurate comparison, the absorption spectrum of N-Ac-0-Me-L-Tyr ethyl ester in methylcyclohexane at 225 K was recorded using the monochromator in the CD instrument so that differences in spectral band widths would not affect the results.
The individual vibronic bands occur at the same wave lengths, and their relative intensities are nearly identical in both the CD and absorption spectrum (less than 10% variation).
N-Stearyl-0-Methyl-L-Tyrosine n-Hexyl Ester-The spectra of N-stearyl-0-methyl-L-tyrosine n-hexyl ester were also examined dissolved in methylcyclohexane at 297 and 225 K. These CD and absorption spectra are nearly identical with those shown for N-Ac-0-Me-L-Tyr ethyl ester in methylcyclohexane in Fig. 3 (see also   measurements were possible for low concentrations of N-AC-L-Tyr ethyl ester (0.4 and 1.9 mM) dissolved in EPA.
At 297 K the CD spectrum is weakly positive (1). Cooling brought out a much more intense, negative CD in the region above 265 nm. The shapes of the 215 and 140 K CD spectra are similar to those of N-Ac-0-;lIe-L-Tyr ethyl ester dissolved in EPA (Fig. 2), but have only about half the rotatory strength (Table  I). For N-AC-L-Tyr ethyl ester in EPA at 140 K the negative CD fine structure occurs at 287 and 281 nm, which are the positions of the major vibronic absorption bands (1). S-Stearyl-L-Tyrosine n-Hezyl Ester-The CD spectrum of this compound is strongly concentration dependent when dissolved in methylcyclohexane.
At 0.2 mM, the CD spectrum has a negative band at 288 nm and positive CD bands at 283 and 276 nm (Fig. 4) dependent of concentration (Fig. 4). In the absorption spectra recorded at low concentrations, the 287.nm shoulder is absent and the vibronic bands are better resolved at 283.1 and 276.7 nm (Fig. 4).
The concentration dependence of the CD and absorption spectra indicates that Wstearyl-L-tyrosine n-hex)-1 ester begins to aggregate at concentrations above 15 pM. Apparently the CD and absorption spectra at 15 pM and below represent the monomeric N-stearyl-L-tyrosine n-hexyl ester dissolved in methylcyclohexane.
The approximate association of AT-stearyl-L-tyrosine TL-hesyl ester was estimated from the increase in absorbance at 287 nm in the more concentrated solutions. This band was assumed to result from the formation of dimers having their O-O ribronic bands shifted to 287 nm. This technique has been extensively used to measure hydrogen bond formation in phenol and p-cresol (18-22). The first experiments were carried out with dioxane and LV, Ndimethylacetamide-compounds which are able to act only as proton acceptors.
The extent of interaction with the phenolic ring of N-stearyl-L-tyrosine n-hexgl ester was determined from the red shift in the absorption spectrum after adding the proton acceptor (Fig. 5) FIG. 5. Effects of l-butanol (left), p-dioxane (center), and N,Ndimethylacetamide (right) upon the absorption spectra of Nstearyl-L-tyrosine n-hexyl ester (9 to 10 ,uM) dissolved in methylcyclohexane.
Solid lines were traced from instrument record to

TABLE II
Effect of hydrogen bond acceptors upon N-stearyl-L-tyrosine n-hexyl ester Acceptor was added to monomeric N-stearyl-L-tyrosine n-hexyl ester (8 to 10 pM) in methylcyclohexane until the phenolic hydroxy group was fully hydrogen bonded, as revealed by the spectral shift (Fig. 5) Fig. 5 compare the spectral effects of dioxane and N, N-dimethylacetamide as proton acceptors for N-stearyl-L-tyrosine n-hexyl ester. Hydrogen bond formation to dioxane causes less red shift and a smaller increase in dipole strength than does bonding to N, N-dimethylacetamide. Furthermore, the equilibrium constant is about 25 times smaller for binding dioxane than for binding N, N-dimethylacetamide (Table III). Alterations in the CD spectra also take place after N-stearyl-L-tyrosine n-hexyl ester is hydrogen bonded to either N, N-dimethylacetamide or dioxane. The red shift and spectral blurring occur to the same extent in the CD spectra (Fig. 6) as in the absorption spectra (Fig. 5). The rotatory strength increases in approximately the same proportion as the increase in dipole strength (Table II).
At much higher concentrations of dioxane or N, N-dimethylacetamide, the rotatory strength decreases appreciably.
Other experiments were carried out with alcohols as hydrogenbonding agents. They differ from the previous compounds in their ability to bond to the phenolic hydroxy group at both the oxygen and hydrogen atom (29). Adding 1-butanol to N-stearyl-1;-tyrosine n-hexyl ester increases the dipole strength by 1370 and red shifts the O-O absorption band by 1 to 3 nm (Fig. 5). The exact value of the shift cannot be measured because the vibronic bands are completely blurred, much more so than with dioxane or N, N-dimethylacetamide.
Alcohols are not intrinsically poor solvents for resolving the major vibronic bands, e.g. O-methyl tyrosine derivatives have their vibronic bands resolved in these solvents (Fig. 2). Apparently the blurring of the N-stearyl-L-tyrosine n-hexyl ester absorption spectrum implies that two or more hydrogen bonded species exist, each having its O-O band shifted by a different amount.
CD spectra recorded after adding butanol to N-stearyl-n-tyrosine n-hexyl ester also reveal blurring of the vibronic bands and a I-to 2-nm red shift.
The  (Fig. 7). Similar changes were observed after adding small amounts of methanol to N-stearyl-L-tyrosine n-hexyl ester dissolved in methylcyclohexane.
In pure butanol the CD spectrum of N-stearyl-L-tyrosine n-hexyl ester is weakly negative from 275 to 290 nm.
To further examine the sites where butanol interacts, CD and absorption spectra were recorded following the addition of buta-no1 to both N-stearyl-0-methyl-L-tyrosine n-hexyl ester and N-stearyl-L-phenylalanine n-hexyl ester. Up to 0.5 M butanol does not blur the vibronic absorption bands of N-stearyl-Omethyl-L-tyrosine n-hexyl ester, although the bands may have been shifted to a slightly shorter wave length (less than 0.2.nm shift).
The dipole strength is essentially unchanged. At low concentrations of butanol the loss of rotatory strength is appreciably less than that observed for the tyrosine compound ( Fig.   7). At 0.5 M butanol, the rotatory strength of N-stearyl-omethyl-L-tyrosine n-hexyl ester declines by 437& which is about the same as observed with the tyrosine derivative.
The possible interactions of butanol with the amide and ester groups were examined with the use of N-stearyl-L-phenylalanine n-hexyl ester dissolved in methylcyclohexane. Measurable changes in the near ultraviolet CD spectrum of this compound began to occur at 0.08 M butanol.
As the butanol concentration was increased further, the CD intensity of the O-O band gradually changed from weakly positive to weakly negative. Even at 1.5 M butanol there was no evidence that the binding sites on N-stearyl-L-phenylalanine n-hexyl ester had been saturated. Evidently butanol interacts at numerous sites on AT-stearyl-Lphenylalanine n-hexyl est,er.

DISCUSSION
Combined studies of tyrosine and O-methyl tyrosine derivatives aid in understanding the spectral properties of the unionized tyrosyl side chain.
The high resolution solution spectra of model compounds (p-methylanisole and p-cresol, Fig. 1 are sharper than those of tyrosine derivatives in polar organic solvents, the dipole strengths are slightly greater for tyrosine derivatives. This difference in behavior results from the phenolic hydroxy group acting as a proton donor in hydrogen bond formation (18). The hydrogen-bonding experiments with N-stearyl-n-tyrosine n-hexyl ester suggest that the stronger hydrogen bonds (measured by the equilibrium constants) may produce greater enhancement of dipole strength. The mechanism involved in this effect has been discussed by other workers (17, 19, 22).
There is only one major spectral difference between O-methyl tyrosine and the unionized tyrosyl side chain; the wave length position of the O-O band of O-methyl tyrosine is much less shifted by hydrogen-bonding solvents than is the case for tyrosine.
In all organic solvents used, the O-O band of O-methyl tyrosine derivatives occurs at 283.5 f 0.5 nm (Figs. 2 and 3). By contrast, the O-O band of N-stearyl-n-tyrosine n-hexyl ester is red shifted 1 to 4 nm by hydrogen-bonding agents added to methylcyclohexane (Fig. 5). The red shift observed under these conditions probably depends largely upon t,he strength of the hydrogen bond (31). Interestingly N, N-dimcthylacetamide, a model compound for the peptide bond, is strongly bound by h'-stearyl-I,-tyrosine n-hexyl ester and causes a 3.5.nm red shift (Table II).
Evidently hydrogen bonding of the tyrosyl hydroxy group to the carbonyl oxygen of a peptide bond should be thermodynamically favorable in proteins and may partially account for the large red shift of the O-O band of some tyrosine residues in proteins (1). For example, in ribonuclease-S one of the 3 tyrosyl residues having its O-O band at 286 nm (32) does have this type of hydrogen bond (see Tyr 92 in Fig. 41 of Reference 33).
In addition to hydrogen bonding, other physical properties of the tyrosyl site may also influence the position of its O-O band in proteins (22, 34). Next we shall examine several factors that influence the near ultraviolet rotatory strength of the N-acyl esters of tyrosine and 0-met'hyl tyrosine.
The largest rotat,ory strength for the momomeric forms occurs in a completely nonpolar solvent, methylcyclohexane (Table I). This solvent permits the strongest interactions between the polar groups of the amino acid portion and the aromatic ring. Perhaps these interactions are sufficiently strong to stabilize mainly a single conformation for these moieties.
The small increase in rotatory strength upon cooling Kstearyl-0-methyl-n-tyrosine n-hexyl ester and N-Ac-0-Me-r-Tyr ethyl ester in methylcyclohesane (Fig. 3) is consistent with this interpretation (see below). Close contact of the aromatic ring with the amide and ester groups would tend to facilitate coupling with their electronic transitions and may thereby produce a large rotatory strength (5). The effects of hydrogen bonding upon rotatory strength were examined by using the red shift of the absorption band to identify when the tyrosyl hydroxy group is hydrogen bonded. These studies, however, are complicated by the occurrence of several other binding sites.
To aid in determining which sites are occu-pied on AT-steargl-L-tyrosine n-hexyl ester, a number of equilibrium constants for hydrogen bonding to model compounds (23-28) have been compared with those of the tyrosine derivative (Table III).
Apparently low concentrations of hydrogen-bonding agents should interact preferentially with the phenolic hydroxy group, though the margin over other binding sites is not always great.
Aft'er adding small amounts of dioxane or &V,A-dimethylacetamide, the CD spectrum of N-stearyl-n-tyrosine n-hexyl ester is red shifted and blurred to the same extent as the absorption spectrum (Figs. 5 and 6). A small increase of rotatory strength occurred in proportion to the increase in dipole strength after hydrogen bonding (Table II).
These findings suggest that the conformation of N-stearyl-L-tyrosine n-hexyl ester is not altered when the phenolic hydroxy group is hydrogen bonded to dioxane or N , N-dimethylacetamide. The relatively minor change in the CD spectrum results because the absorption spectrum is altered.
Contrary to the results with dioxane and N,N-dimethylacetamide, adding even small concentrations of butanol or methanol causes the rotat,ory strength of N-stearylln-tyrosine n-hexyl ester to decrease by half.
Both the CD and absorption alterations have approximately the same concentration dependence (half-maximal effect at 30 m&I butanol, Fig. 7). The apparent equilibrium constant for this interaction agrees well with the values published for alcohols hydrogen bonding to the phenolic hydroxy group (Table III).
If each hydrogenbonding site acts independently, butanol should bind less well to the other sites on N-stearyl-n-tyrosine n-hexyl ester. The second strongest binding site for butanol is expected to be the amide oxygen atom.
The equilibrium constants predict that about 357; of the amide oxygens should be hydrogen bonded when the phenolic hydroxy group is essentially fully hydrogen bonded (0.1 RI butanol).
At 0.5 M but'anol, about 750/ of the amide osygens should be hydrogen bonded.
Surprisingly, the rotatory strength of N-stearyl-n-tyrosine n-hexyl ester does not decrease any further when the butanol concentration is raised from 0.1 to 0.5 JI (Fig. 7).
In contrast, the Kstearyl n-hexyl esters of phenylalanine and O-methyl tyrosine do not have a plateau region. For these compounds, the rotatory strengt'h is less altered at low concentrations of but,anol than is the case with the tyrosine compound (Fig. 7). Ss the butanol concentration is increased to 0.5 M and above, the rotatory strengths of the O-methyl tyrosine and phenglalanine derivatives continue to change. Apparently at these higher concentrations butanol interacts extensively with the amide and ester groups of these compounds lacking a phenolic hydroxy group.
For N-stearyl-n-tyrosine n-hexgl ester, the large initial loss of rotatory strength seems in some way to involve the interaction of butanol with the phenolic hydroxy group.
The structures of the alcohol-phenol complexes are not well understood, in part because their hydroxy groups can function as both prot'on donors and acceptors.
When phenol donates a proton to the osygen atom of an alcohol, the alcohol becomes a stronger lproton donor and the phenolic oxygen becomes a better proton acceptor (29). Thus the initial hydrogen bond potentiates additional hydrogen bonds with other alcohol molecules, leading to phenol-polymeric alcohol complexes (29). Undoubtedly a number of different tyrosyl-alcohol complexes are formed, which account for alcohols blurring the absorption spectra (Fig. 5). ,1n examination of molecular models revealed a group of complexes that is especially interesting. As few as 2 or 3 alcohol molecules joined by hydrogen bonds can simultaneously hydrogcn bond to both the hydroxy group and the amide oxygen atom of ~-atearvl-L-tvrosirlc n-hex\-1 ester.* A complex of this type would explain the large initial decrease in rotatory strength at low butanol concentrations and the absence of any further effects between 0.1 and 0.5 M butanol.
In any case, it seems highly probable that the binding of even low concentrations of alcohols leads to an altered conformation for A-stearyl-L-tyrosine n-hesyl ester.
Even with pure alcoholic solvents, the conformations of these tyrosine derivatives do not seem to be much affect,ed by the phenolic hydroxy group donating a proton to form a hydrogen bond with the solvent. l\lajor differences between the CD spectra of tyrosine and O-methyl tyrosine compounds were observed only whru the tyrosine derivat,ives aggregated (Fig. 4). The 1)henolic hydroxy group in tvrosine derivatives greatly increases the tendency to self associate in organic solvents.
The aggregation of N-stearyl-L-tyrosinc rL-hesyl ester in methylcyclohexalle apparently reveals the interacting groups.
In contrast to p-cl-es01 aggregation, au interaction between the tyrosyl hydrosy groups does not seem to occur.
First of all, the tyrosine interaction is much stronger than that of p-cresol (Table III).
Secondly, the aggregation of N-stear~l~L-t~rosinc n-hexgl ester in methylcyclohexanc causes a 4.11111 red shift (Fig. 4) instead of the short wave length shift observed in p-cresol aggregates (28). Perusal of the hydrogen bond rquilibrium constants (Table III) reveals that the strongest possible interactions occur between phenolic hydroxy groups and amide osv"en atoms.
Examining models of n'-stearyl-L-tyro-" r, sine n-hexyl ester showed that a dimer can be formed having the hydrosy group of each tyrosine residue hydrogen bonded to the amide oxygen atom of the other residue.2 In this dimer most of the two aromatic rings are stacked in van der Waal contact. Evidentiy this conformation has sufficient interactions to account for the large equilibrium constant and the altered CD spectrum of the aggregate.
At high concentrations, long chain polymers of S-st,earyl-L-tyrosine n-hexyl ester may be formed by having the h\-droxy group of one residue hydrogen bonded to t'he amide oxygen atom of the following residue.
As our final point, we consider the effect of temperature upon the CD spectrum of S-Ac-L-Tyr ethyl ester and X-Sc-0->le-L-Tyr ethyl ester dissolved in EPA.
The measured CD spectrum is the population-weighted average resulting from the various conformers existing in equilibria (35). At room temperature, the tliat,ribution of the various conformers is such that their population-weighted average gives a very small rotatory 2 The hydrogen atom of the tyrosyl hydroxy group remains in the plane of the aromatic ring.

strength.
Probably this results because the number of conformers with positive CD spectra is approximately equal to the number with negative CD spectra, giving nearly complete cancellation.
Cooling EPA solutions of X-Ac-0-hle-r-'l'yr ethyl ester and N-dc-L-T)-r ethyl ester from 297 K to 140 K causes the rotatory strength to become strongly negative, especially for h'-Ac-0-hle-L-Tyr ethyl ester. Evidently cooling shifts the equilibria in favor of conformers having negative Cl) spectra, i.e. conformational motility (36) exists. The large enhancement of rotatory strength upon cooling N-AC-0-Me-L-Tyr ethyl ester (Fig. 2) is similar to that observed previously with nonrigid tryptophan and phenylalanine derivatives (7, 37). hpparcntly the noncyclic derivatives of all these aromatic amino acids may have extensive motility in EPA. Low temperature CD measurements arc also possible for proteins that dissolve in a water-glycerol solvent without having their native conformation altered (32,38). Thus variable temperature CD spectra provide a means to detect motility-of the aromatic amino acid side chains of proteins.
In fact, this technique has already revealed some motility in the tryptophanyl side chains of carboxypeptidase A (38).