correlation between computed conformational properties of cytochrome c peptides and their...

Correlation Between Computed Conformational Properties of Cytochrome c

Peptides and Their Antigenicity in a T-Lymphocyte Proliferation Assay

MAX VASQUEZ, Baker Laboratory of C h e ~ t r y , Cornell University, Ithaca, New York 14853-1301; MATTHEW R. PINCUS,

Department of Pathology, New York University Medical Center, New York, New York 10016; and HAROLD A. SCHERAGA, *Baker

Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301

Synopsis

By means of conformational energy calculations, we previously showed that the antigenic strength of a series of oligopeptides (derived from the carboxyl terminal sequence of cytochrome c) in a T-lymphocyte proliferation assay depends on their ability to adopt the a-helix conformation. Using experimentally determined statistical weights (within the framework of the Zimm-Bragg theory for the helix-coil transition), here we present a simple free energy analysis of the ability of these peptides to adopt the a-helix conformation in water. The experimental statistical weights have been modified to include the effect of long-range charge-dipole interactions on helix stability. We find that there is a close correlation between the tendency of a peptide to adopt the a-helix conformation and its ability to stimulate antigen-primed T cells. The shortest peptide with a tendency to adopt the a-helix conformation is also the shortest one that exhibits antigenic activity. The rapid and simple method presented here can thus be used to predict relative antigenicities for different peptides derived from cytochrome c.

INTRODUCTION

Conformational energy calculations have provided an understanding of how interatomic interactions lead to the observed behavior of a variety of biological macromolecules.' It has thus been possible, in many cases, to correlate biological activity with molecular structure. For example, in a recent appli- cation of such computations,2 it was shown that the ability of different peptides derived from the carboxyl terminus of cytochrome c to stimulate antigen-primed T cells in a T-lymphocyte proliferation a s s a ~ ~ , ~ seems to depend on whether or not they adopt the a-helix conformation. In related experiments, it was demonstrated that T cells stimulated by pigeon cytochrome c could be fully stimulated by the carboxyl terminal sequence of this protein, residues 81-104,3 and that most of the antigenic activity resides in residues 87-104.4 The sequence of these residues (87-104) in pigeon cytochrome c is shown in Table I. These experimental studies have further shown that (a) deletion of Ala 103 results in enhanced antigenic activity; (b) the Ia

*To whom requests for reprints should be addressed.

Biopolymers, Vol. 26, 373-386 (1987) 8 1987 John Wiley & Sons, Inc. CCC 0006-3525/87/030373-14$04.00

374 ANTIGEN STRUCTURE AND T-CELL ACTIVATION

TABLE I Amino Acid Sequence of the C-Terminal Region

of Cytochrome c from Pigeon’

87 88 89 90 91 92 93 94 95 LY5 LYS Ala Glu k g Ala ASP Leu Ile

96 97 98 99 100 101 102 103 104 Ala TYr Leu LYS Gln Ala Thr Ala LYS

‘Ala 103 is deleted from the sequence of moth cytochrome c, and in the des-Ala 103-pigeon antigen. The moth sequence also contains substitutions2 of Ala and Am for Lys and Ma, respectively, at positions 88 and 89.

molecule on a macrophage recognizes the terminal Lys 104(103); (c) the T-cell receptor most probably recognizes the r-amino group of Lys 99; and (d) residues 87-98 probably do not interact with either the T-cell receptor or the macrophage Ia molecule, but are necessary for the expression of antigenic activity.

Using conformational energy calculations,2 we showed that residues 87-98 strongly promote the a-helix conformation among residues 99 to 103, and that residues 87-98 most likely serve to stabilize this conformation necessary for antigenic activity. We further found the following: (a) The peptide 99-103 from the des-Ala 103 pigeon sequence has no preferred structure; it is thus a statistical coil and therefore should not be a good antigen even though it contains the antigen recognition sites. Recent experimental results have confirmed this ~rediction.~ @) The peptide 94-103 from the des-Ala 103 pigeon sequence, which contains the a-helix nucleating segment 94-98, does exhibit a preference to adopt the a-helix conformation and hence should manifest antigenic activity. This predicted result was corroborated by a direct test in which this molecule was synthesized and shown to stimulate pigeon antigen- (81-104)-primed T cells.4 (c) The sequence 91-93 tends to destabilize a-helices; we therefore predicted that this subsegment could be deleted from the moth antigen (86-103) to yield the “splice” sequence (86-90)-(94-103), without loss of antigenicity, as was indeed found to be the case.4 These calculations thus seem to establish a correlation between peptide

conformation (a-helix) and antigenic activity. However, such calculations are quite time-consuming, becoming significantly longer as the length of the oligopeptide under study increases. Furthermore, thus far they have been concerned only with conformational energy, i.e., only the global (energy) minima were sought to detennine whether a peptide showed a preference for a given conformation; entropy and, therefore free energy, considerations were omitted.

In the present treatment, we use a statistical mechanical method for rapid evaluation of the helix content of a given peptide based on free energy considerations. This method provides a quantitative estimate not only of the helix content of a given peptide but also of the average tendency of any given residue or group of residues in a peptide to adopt the a-helix conformation. We can thus study the effects of adding different residues to the basic sequence 99-103 (which contains the proposed antigen recognition site) and determine the minimal-length peptide that will manifest significant antigenic activity in a rapid and straightforward way.

VASQUEZ, PINCUS, AND SCHERAGA 375

Statistical mechanical theories for the helix-coil transition in biopolymers have been formulated since the late 1950~.~ However, only later was it possible to develop techniques to determine both theore t i~a l~ ,~ and experimental'-'' values for the parameters introduced in those theoretical formulations. In particular, experimental values of the parameters s and u of the original Zimm-Bragg formulation5 have been obtained in this l a b o r a t ~ r y ~ ~ ~ ' for most of the naturally occurring amino acids. Preliminary sets of these parameters have been used by Holtzer, Skolnick, and c~llaborators~'-'~ in the study of thermal transitions of the muscle protein a-tropomyosin. These authors have also developed a treatment for the consideration of interactions between separate polypeptide chains undergoing helix-coil transitions. An earlier ap- plication of the Zimm-Bragg theory was carried out for the estimation of the relative stability of a-helix segments in denatured proteins.14-16

Recently17 we have extended the Zimm-Bragg model5 to include long-range charge-dipole electrostatic effects on the experimentally determined helix-coil transition parameters" u and s. These long-range effects, also parameterized experimentally, have been incorporated into the original simple formulation and applied to the computation of helix-probability profiles for a number of specific-sequence polypeptides (such as the S peptide of ribonuclease A) and proteins of known structure, with reasonably good agreement between predicted helical regions and those found e~perimental1y.l~ We now apply these parameters and the modified theory to the correlation of helix structure with the antigenicity of the cytochrome c carboxyl-terminal oligopeptides.

THEORETICAL MODEL

The theory and parameters used in the present computations are described in the accompanying paper.17 The data are presented as plots of PH( z ) vs z, or values of PH(A) for given sequences, where these quantities were defined previo~sly.'~

The computations were performed first on the sequence 99-103 of des-Ala 103 pigeon cytochrome c, since this peptide contains the antigen recognition site. Residues were added sequentially (starting from position 98) onto the amino terminus of a particular peptide. The results of the calculations are compared with those from a T-lymphocyte proliferation assay obtained recently by Schwartz et al.18

In the present study, the helix stabilities of the different peptides are calculated at three temperatures, viz., 0, 25, and 37°C. The results of the calculations at 0°C are the ones used to compare with experimental data because the peptides are most highly structured at low tem~erature'~ and because there is good evidence that the antigenic peptides become highly structured in a nonpolar membrane environment when interacting with surface membrane receptors as exist on T ~ e l l s . ~ ? ' ~

RESULTS AND DISCUSSION

Helix Probability Proflles

The computed results for the average helix content, defined as the average (PH) of the PH(i)s [eq. (3) of Ref. 171, of peptides i to 103 (for 86 < i < 100) are presented in Fig. 1. As found previously,2 the pentapeptide 99-103 is not


0.3 I I I I I 1 ‘ 1 1 ~ I I I I , I

0.015

-

0.005

90 95 100- Fragment i - to-103

0.2 -

0.1 - -

-

-0 I I J

85 90 95 I00 Frogment i - t o - 1 0 3

Fig. 1. Average value of PH(z) of Eq. (3) of Ref. 17 for peptides 87-103 to 99-103 of des-Ma 103 pigeon cytochrome c at temperatures of 0 (A), 25 (o), and 37°C (0). Inset: same calculation for poly(L-alanine) chains of equivalent length.

a-helical. A significant increase in this quantity is observed when residues 95, 94, and 93 are added to the N-terminus of the 96-103 peptide fragment. At O’C, peptides 92-103 and longer show a small decrease in the average helix probability upon addition of residues at their N-termini. Similar properties for poly(L-Ala) chains, with the same lengths as the peptides considered above, were also computed (inset of Fig. 1). The latter results show a pure length effect in the average helix probability; i.e., (PH) increases with increasing chain length. The average probability for poly(L-Ala) is always lower than that for the cytochrome c peptide of corresponding length. Comparison of the poly(L-Ala) curves with the other curves of Fig. 1 suggests that the behavior predicted for the fragments derived from cytochrome c is due primarily to a sequence rather than to a length effect. I t should be noted that the parameters used to compute the data of Fig. 1, and all subsequent figures, pertain to amino acid residues in water at neutral pH (so that Glu, Asp, Lys, and Arg side chains should be considered charged), whereas our earlier conformational energy computations on cytochrome c peptides2 considered the ionizable residues as uncharged and the “medium” (vacuum) as a nonpolar one.

Helix probability profiles for the i-to-103 peptides are presented in Fig. 2. Since experimental results strongly suggest that Lys 99 is involved directly in peptide-receptor intera~tions,~ special attention is given to the region surrounding this residue. Several well-defined jumps in the helix probabilities of Lys 99 and of its nearest neighbors (in sequence) can be seen in Fig. 2. The first occurs between the 98-103 and the 97-103 fragments, another between the 96-103 and the 95-103 polypeptides, and a very large one between 94-103 and 93-103 upon addition of the negatively charged Asp 93 to the N-terminus.


Residue i in Fragment i - t o - 1 0 3

Fig. 2. Helix-probability profiles, PH(i ) vs i [Eq. (3) of Ref. 171, for the z-to-103 peptides, calculated at 0°C.

The average helix content ( PH) for each peptide, as determined for Fig. 1, can be used as a “cutoff” value for deciding whether or not a residue like Lys 99 exhibits a tendency to exist in the a-helix c~nformation.’~ Comparison of PH(99) for each of the peptides in Fig. 2 with the values of (PH) for each peptide in Fig. 1 reveals that Lys 99 and surrounding residues become “helical” beginning with the peptide 97-103. This tendency becomes increas- ingly strong as more residues in the sequence are added to the amino terminus. We therefore predict that the shortest peptide that should exhibit antigenicity is 97-103.

In Fig. 2, it may be observed that addition of residues toward the amino terminus beyond residue 93 continues to affect the values of PH for residues 99 and its immediate neighbors, but in smaller increments or decrements. In fact, all the peptides that contain the sequence 93-103 form a discrete “cluster” of sequences (Fig. 2) having similar values of PH(99). Thus, we predict that peptides that contain the sequence 93-103 should exhibit the strongest antigenicity. The effect of residue 93 (aspartic acid) may be to promote helix formation by introducing a negative charge near the amino- terminus of the helix.I7

In a prior publication,2 we showed that the sequence 94-103 (from moth cytochrome c ) formed a stable a-helix but that the preceding tripeptide Arg 91-Asp 93 showed little preference to exist as an a-helix. We further found that the moth sequence Lys 87-Glu 90 spliced with Leu 94-Lys 103 showed a strong helix preference. In the case of Arg 91-Asp 93, side chain-side chain interactions between Arg 91 and Asp 93 caused this peptide to be nonhelical while, in the case of Lys 86(87)-Glu 90, the side chain-side chain interactions between the Lys residues and Glu 90 greatly stabilized the a-helix. We


0.3

0.2

P,, (i)

0. I

n

I

-6-6- - 85 90 95 100 I05

Residue Number (i) Fig. 3. Helix probability profiles, PH(i) vs i [Eq. (3) of Ref. 171, for the splice sequence from

moth cytochrome c. The moth sequence 94-103 is shown for comparison. Calculations pertain to 0°C.

therefore predicted that the peptide 94-103 should be antigenic and that the splice sequence, in which the segment 86(87)-90 is attached directly to 94-103, should be strongly antigenic. Both predictions were confirmed experimentally.* In Fig. 3, the helix probability profile for the splice sequence (86-90)-(94-103) is presented together with that for the 94-103 sequence. The presence of Lys 86-Glu 90 substantially increases PH(99) (and PH for all other residues) in the splice sequence relative to that in the 94-103 sequence, consistent with the prior calculations.2

Helix Probabilities for Subsequences

Since the biologically active conformation of each peptide studied is likely to depend on the state of more than one residue simultaneously, helical probabilities for groups of residues within each polypeptide were also computed (Fig. 4). Joint helical probabilities were calculated for the groups of residues 98-100 and 98-101. The results obtained are qualitatively similar for both sets: there is a small tendency for a-helix formation in peptides 97-103 and shorter, but this tendency is greatly enhanced upon the addition of three more residues, viz., 95, 94, and 93. Addition of more amino acid residues (92, 91, etc.) causes slight increases or decreases in helical probabilities. Because Lys 103 is implicated as being involved in interactions with the macrophage Ia molecule, we also computed the probabilities of finding the two Lys residues, 99 and 103, simultaneously in a helical state, as well as the corresponding quantity for the whole segment 99-103 (which contains the proposed antigenic determinants). The results, also shown in Fig. 4, for the 99 and 103


PH

85 90 95 Fragment i- t o - 103

I00

Fig. 4. Values of P,(A) of Eq. (4) of Ref. 17 for the sets A = {98,99, 100}, {98,99, 100, lOl}, {99, 100, 101, 102, 103}, and (99, 103), from top to bottom, respectively (the last two sets corresponding to the same curve) in peptides i to 103. Inset: same calculation for poly(L-alanine) Chains.

residues (which are similar to those for the 99-103 set of residues) exhibit an increase in helix tendency when we go from peptide 96-103 to 93-103. This increase occurs mainly upon addition of Asp 93. Similar curves were computed for poly(L-alanine) chains (inset in Fig. 4). The conclusion drawn by compar- ing these curves with the corresponding ones for fragments of cytochrome c is that the sharp increases, observed at the specific locations in the cytochrome c peptides as discussed above, are due to true sequence effects. It is noteworthy that, in all peptide segments studied, PH(103) is always low and is in fact lower than ( PH) for each peptide. This result occurs in part because Lys 103 is an end residue and is less constrained by the propagating helix. However, it seems clear that the conformational requirements for this residue are less stringent than for Lys 99. It may also be noted that distinct "dips" in the helix probability profile occur at residue 91, an Arg residue. This effect is consistent with the results of the calculations described above,' and is due to the presence of a positive charge near the amino-terminus of the helix; it may partly explain why the splice sequence, in which this residue is absent, serves as a more effective antigen. While Asp is a helix-breaking it is often found at the beginning of a-helices because its negatively charged side chain stabilizes the l79'O

T-cell Response to Synthesized Cytochrome c Peptides

A number of different peptides have been synthesized18 and used to chal- lenge T cells from BIO.A mice lymph nodes, primed with pigeon cytochrome c peptide 81-104. The dose response curves for different concentrations of each peptide are parallel, indicating similar mechanisms of stimulation.18 In these


TABLE I1 Antigenicities of Cytochrome c Peptides of Increasing Chain Length” and Predicted Helical Indices

Concentration to

of 1 x lo5 cpm Stimulate Uptake (pH)

Relative (PM) Shiftb 0°C 25OC 37OC

~ ~~ ~

81-104 (pigeon) 81-103 (moth) 93-103 94- 103 95-103 96-103 97-103 98- 103 99- 103 Splice (87-90)-(94-

0.003 0.01 1 0.216 0.120 0.170

15.0 104.0

a3 -103) 0.106

0.3 1.0

20 11 15.5

1400 9500

a3

10

17 14 26 6 5 2 2 0.6 0.2 9

4 4

19 4 4 2 2 0.6 0.2 4

2 2

15 4 3 1 2 0.6 0.2 2

~ ~~ ~~ ~~~~~

‘See Ref. 18 and R. H. Schwartz, personal communication. bData in previous column ace divided by 0.011.

experiments, T lymphocytes (harvested from BIO.A mouse lymph nodes) were incubated with [3H]thymidine and antigen, and the number of counts of [ 3H]thymidine incorporated was measured. At high antigen concentrations, the response diminishes, possibly because of activation of T-suppressor cells. To obtain a quantitative measure of antigen strength, therefore, the concentration of antigen needed to obtain a fixed response was determined for each antigen. The response (i.e., cpm of [3H]thymidine incorporated) was selected as 100,OOO cpm, a value close to that at saturation. Table 11 is a summary of the results obtained with each peptide (Ref. 18; R. H. Schwartz, personal communication), together with the average calculated helix probability and the helix probability for Lys 99. These data are plotted in Fig. 5, where the negative of the natural logarithm of the concentration of peptide needed for 100,OOO cpm incorporation is plotted vs chain length. The negative of the natural logarithm of concentration is used as a quantity assumed to be roughly proportional to a free energy of “antigen-receptor” binding.

Comparison of Computed Helix Content with Experimental Results

F’rom Table 11, and from a comparison of Figs. 1 and 2 (helix probability profiles) with Fig. 5 (experimental results), it may be seen that a direct correspondance between helix probability and antigenicity exists. The first peptide to exhibit antigenicity is the 97-103 sequence (Fig. 5), as predicted and discussed above. Discrete increases in average helix probability ( PH), and in individual residue helix probabilities PH( i), occur upon addition of residue 97 and then again at residue 95 (Table II, 0°C data, and Figs. 1 and 2), as mentioned above. As may be seen in Table I1 (OOC data), ( PH) increases by a factor of 2.5 when residue 95 is added to the sequence. Correspondingly, in the


2'o: /*'*'*

0.0

-Qn C

-2.0

I 1 I I 1 I I

9 5

-4.0 -

I00 F r o g m e n t i - t o - I 0 3

Fig. 5. Plot of negative (natural) logarithm of antigen concentration (in p M ) needed to obtain 1 x lo5 cpm in T-lymphocyte proliferation assay for cytochrome c peptides of different chain lengths.

L I 1 I I 1 I I J I00 9 5

F r o g m e n t i - t o - I 0 3

Fig. 5. Plot of negative (natural) logarithm of antigen concentration (in p M ) needed to obtain 1 x lo5 cpm in T-lymphocyte proliferation assay for cytochrome c peptides of different chain lengths.

experimental curve, addition of residue 95 causes a significant increase in antigenicity. An additional structural basis for this observation is discussed further in the next section. A sharp increase again is observed upon addition of Asp 93, an effect discussed in the preceding section. The values for PH(i) and, in particular, for PH(99), appear to plateau upon addition of residue 93, although a further addition of residues produces small changes in this value back to residue 87 (Fig. 2). These results correspond to the increases in antigenicity up to and including addition of residue 94, after which there is a slight decrease at Asp 93. The antigenicity increases again (not shown here) somewhat upon addition of the remaining residues back to Lys 86.3,4,18 Thus, there is an overall correlation between a-helicity and antigenicity for the peptides studied here.

It should be noted, however, that the orders of magnitude of the relative changes obtained in the calculations and in the immunochemical experiments are different. Thus, for example, from Table I1 and Fig. 5, there is approximately a 100-fold increase in antigenicity of the 94-103 peptide relative to that of the 96-103 peptide. The helix probability increases by approximately a factor of 3. Further, there is a minor discrepancy between helicity and antigenicity for the peptides 93-103 and 94-103, the latter having been found to be slightly less than two times stronger as an antigen. The experimental result was obtained with A.E7 T-cell clones. However, the reverse pattern is found for another T-cell clone (2B4) which is stimulated 4-8 times more strongly by the 93-103 sequence than by the 94-103 sequence.18 Our calculations predict an increase in helix content when residue 93 alone is added, consistent with the results from the 2B4 clone.


Experimental Determinations of Helix Content

The correspondence between helix content and antigenicity receives further support from CD experiments" in which the helix content at 37"C, pH 7.4, was observed in a mixed solvent, trifluoroethanol (TFE) in water, for each of several cytochrome c antigenic peptides, viz., peptides 96 to 103, 95 to 103, 94 to 103, and 93 to 103 (with a Glu residue in place of Asp at position 93) and the moth splice sequence (86 to 90)-(94 to 103). While little or no helix was observed in pure water at 37°C for these peptides, increasing helix content was observed as the concentration of TFE was increased." A t concentrations of 50% TFE in water, the helix content of each peptide appeared to plateau at approximately 14% for the splice sequence, 10% for the 93 to 103 sequence, 4-68 for the 94 to 103 sequence, 3% for the 95 to 103 sequence, and 2% for the 96 to 103 sequence. The helix contents of each of these peptides therefore correlate at least roughly with their antigenic strengths. As seen in Table 11, at 37°C in pure water, helix contents are expected to be low, as indeed was found experimentally. Since, however, the antigenic peptides very likely interact with the T-cell receptor and Ia molecule at a membrane surface, the environment in which the interactions occur is not a purely aqueous one, and is more likely simulated by the addition of the helix-forming solvent TFE.

Comparison of the computed helix contents in Table 11, which pertain to pure water, with the helix contents observed in TFE-water mixtures is not strictly valid. It has been demonstrated that mixed nonaqueous solvents can substantially alter the values of u and s for different amino acids and can change their relative helix-forming tendencies.21 In the current studies, however, a substantial fraction (50%) of the mixed solvent is aqueous. Important hydrophobic and electrostatic interactions would not be expected to be disrupted as they are in a completely nonaqueous environment.21

It is clear that experimental and theoretical trends for the peptides studied here are quite similar. For example, the ratio of the predicted helix content of the 93 to 103 sequence to that of the 94 to 103 sequence from Table I1 at any temperature is 3.5 to 4, and that observed in 50% TFE in water, is 2.5. The exception to this correspondence is the splice sequence whose helix content has been underestimated because the i to i + 4 electrostatic interactions between the side chains of Lys 86 and Glu 90 have been omitted in the present theoretical treatment (see Ref. 17)

Effects of Substitutions at Position 95

From Figs. 1 and 2, it may be seen that Ile 95 strongly promotes the a-helix conformation [(pH) increases significantly and the values of PH(i) also increase when this residue is added; see, e.g., pH(99)]. F'rom Fig. 5, it can be seen that peptide antigenicity correspondingly increases significantly upon addition of Ile 95 to the 96-103 peptide. In the a-helix conformation of the antigenic peptide 87-103 (104) (Table I), Ile 95 and Lys 99 lie near one another (the C=O group of Ile 95 hydrogen bonds to the NH group of Lys 99, and the side-chain atoms of both residues can approach one another), as can be seen in Fig. 6, which is a model of the computed lowest energy (a-helical) structure for the sequence 87-103.' Thus, Ile 95 may be critical both for maintaining peptide structure and for

interacting with Lys 99 and/or the T-cell receptor. (See Fig. 1 of Ref. 22 for a


Fig. 6. Model of the calculated lowest energy (a-helical) structure (Ref. 2 and M. R. Pincus & H. A. Scheraga, unpublished data) of the cytochrome c antigenic peptide Lys 87-Lys 103. The proximity of the side chains of Ile 95, Lys 99, and Lys 103 at the bottom side of the figure should be noted. They all point in the same direction, which is opposite to that of the nonpolar side chains of Leu 94 to Leu 98.

drawing that shows how a nonpolar side chain can interact with the nonpolar part of a polar side chain.) Recently, a number of cytochrome c peptides 95-103 with different amino acids at position 95 have been synthesized and tested for antigenicity against T-cells primed with the pigeon sequence 81-104.'* The most dramatic effects were found when amino acids with long charged side chains such as Glu and Lys were substituted for Ile 95. The presence of Glu reduced antigenicity by a factor of over 50 while Lys reduced it18 by a factor of over 200. Substitution of Lys for Ile 95 reduces both (PH) and PA(98-100) by a factor of 8. Thus, in the case of the positively charged residue, loss of antigenicity correlates with helix destabilization. However, the substitution of Glu for Ile 95 increased the value of ( PH) slightly, as observed in Table I11 and Figure 7. This result suggests that the length and charge of a

TABLE I11 Effect of Amino Acid Substitution at Residue X

at Position 95 for Peptide X-96 to 103

X -In cn (PH) x lOOb

Ile Val Phe Met Gln Glu LYS

2.36 1.87 1.52 1.27 1.14

- 1.12 - 1.26

~

4.8 1.5 2.4 4.8 3.1 5.4 0.6

*C is the micromolar concentration necessary to obtain 100,OOO cpm upon incorporation of

b(PH) is the average helix content calculated at 0°C. tritium-labeled thymidine in an immunogenic assay."


0.10

P,(i)

0.05

0

Residue Number (i) Fig. 7. Helix probability profiles, PH( i) vs z [Eq. (3) of Ref. 171, for peptides X-96 to 103 with

X = Glu (A), Met (0), Ile (0), Lys (a), and Glu (0). Calculations pertain to 0°C.

side chain at position 95 is of critical importance and may overcome its conformational effects.23 A possible explanation is the proximity of the side chain of Glu 95 (or Lys 95) to that of Lys 99, in the a-helical conformation of the peptide, which may cause interference with receptor binding and may interact directly with the side chain of Lys 99 so as to prevent proper orientation for binding to the receptor.

It is noteworthy that changes in other regions of the antigenic peptide segment 88-98, such as modification of Tyr 974 and deletion of residues 91-93 as in the splice ~equence,~1~ cause little or no change in antigenicity, in contrast to the results obtained with substitutions for Ile 95. Since residue 95 is close to Lys 99 in an a-helix (see Fig. 6), these results further support the idea that the active antigenic conformation is an a-helix and that these two residues lie near one another.

Conformation of the Antigenic Peptide

A calculation of the most likely structure of the fragment 94-1032 and examination of the structure of cytochrome c from tuna, as determined by x-ray crystallography,24* 25 reveal the following: (a) the entire carboxyl-terminal sequence 87-103 is an a-helix; (b) part of the hydrophobic face (residues 94-98) of the helix formed by residues 87-103 is buried against the bulk of the protein; (c) residue 99 (Lys in both tuna and pigeon) is exposed on the surface of the protein; and (d) residue 95 (Val in tuna, Ile in pigeon) is not buried, and its side chain points in the same direction as that of Lys 99 (see Fig. 6).


It thus appears that the structure recognized, and probably also induced, by the receptors on the T cells is an a-helix; this helix should have a residue with a small uncharged side chain at position 95, i.e., on the same face as Lys 99 (see Fig. 6), with the latter residue implicated in binding.4 It is interesting that the nonpolar side chains of residues 94,97, and 98 all form a hydrophobic surface of the molecule that is on the opposite side of the helix from that of the polar side chains, especially Lys 99 and Lys 103 in the des-Ma 103 pigeon sequence3p4 (assuming that Lys 103 occurs in a helical state2). Lys 99 and Lys 103 have side chains that point in the same direction and are close to one another (see Fig. 6), possibly implying that the T-cell receptor and the Ia molecule may be brought into close proximity with one another during the immune response. One factor thought to stabilize a-helices is the “amphipathic” side-chain arrangement in which side chains of a hydrophobic sequence point in the opposite direction from those of a neighboring hydrophilic ~equence.~‘?~~ The amphipathic nature of the helix can be seen clearly in Fig. 6. It is interesting that, in a number of other antigenic peptides in different systems involving cell-mediated immunity, a recurring feature of the antigenic determinants was the presence of an amphipathic sequence.28

The occurrence and significance of specific patterns of hydrophobicity in a-helices, in cases involving the interaction of flexible polypeptides with surfaces, such as phospholipid surfaces, membranes, and receptors, have been documented and successfully exploited in the design of active analogs by Kaiser, Kkzdy, and ~ol labora tors .~-~~ Also, Mattice and co-workers have developed an extension of the Zimm-Bragg theory to evaluate helical tendencies of peptides interacting with zwitterionic phospholipid^.^^ It should be pointed out, though, that some but not all of the general findings of these authors could apply to the present case or other cases of interest in immunol- ogy; the nature of the interaction between the antigen (or antigen analog) and the T-cell receptor is expected to have a more specific character.

CONCLUSIONS

Evidence has been presented in support of a simple structure-activity hypothesis for a series of peptides derived from cytochrome c. The experimentally determined values of u and s’, appropriately modified to include the effects of long-range electrostatic interaction^'^ used in the framework of the Zimm-Bragg formulation of the helix-coil transition, have enabled us to detect trends and correlations between a-helicity and antigenic activity for intermediate-sized oligopeptides. These same u and s’ parameters have also provided the empirical basis, in related work, for an understanding of conformational transitions in a-trop~myosin.’~

In this work, the Zimm-Bragg model (with the pertinent parameterization) has offered information leading to the delineation of helical segments within given polypeptide chains. The structural information, together with simple geometrical considerations, provide a sufficient basis for the formulation of a satisfactory interpretation of a series of immunochemical experiments.

We thank Dr. R. H. Schwartz and his co-workers of the National Institutes of Health for making their immunological data available to us prior to publication, and for their generous


permision to use their data for comparison of our calculated results with their experimental findings.

This work was supported by research grants from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases of the National Institutes of Health (AM-O8465), and from the National Science Foundation (DMB84-01811).

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Received April 28,1986 Accepted August 28,1986

correlation between computed conformational properties of cytochrome c peptides and their...

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