J. Phys. Chem. 1988, 92,4759-4764
4759
Poly(DL-proline), a Synthetic Polypeptide Behaving as an Ion Channel across Membranes: Conformational Studies on Ion Complexes of the Tetramer Boc(pPro-L-Pro),OCH, P. De Santis,* A. Palleschi, M. Savino, and A. Scipioni Dipartimento di Chimica, Universitd di Roma, P.za A. Mor0 5, Rome, Italy, and Centro per lo Studio degli Acidi Nucleici del CNR, Dipartimento di Genetica e Biologia Molecolare. Universitd di Roma, P.za A . Mor0 5, Rome, Italy (Received: July 14, 1987: In Final Form: February 17, 1988)
Theoretical and experimental conformational analyses by circular dichroism and NMR spectroscopy of poly(DL-proline) and its tetrameric derivative are reported. The tetramer Boc(D-Pro-L-Pro)zOCH,conformations were investigated in different solvents and in the presence of Ba2+and Ca2+ions to explain the ability of poly(m-proline) to increase the ion permeability across bilayer membranes. Both theoretical and experimental data indicate the formation of dimeric and monomeric complexes between tetramer and cations in solution when the tetramer is in the all-trans conformation of the peptide bonds. The results of this analysis allow confirmation of the channel model, previously proposed on the basis of the phenomenological behavior of poly(DL-proline) in membranes.
Introduction
Conformational Analysis
Naturally occurring peptides and depsipeptides are known to activate the ion passage across biological as well as synthetic membranes. The mechanisms proposed involve the formation of stationary or transient ion complexes, the first operating as ion carriers and the others as ion channels across membranes. Several years ago, we investigated the conformational stability of polypeptide chains with alternating configurations and found such a feature to be associated with the stabilization of folded conformations where the carbonyl groups face in and the side chains face In the case of poly(DL-proline) with all-trans peptide groups, the only sterically possible conformation is represented by a flat helix characterized by an inner channel, where the carbonyl groups point, suitable for the ion passage (see Figure 1). This model mimics the well-known /3-helix first proposed by Urry4 for gramicidin A at the maximum libration amplitude of the peptide groupss W e have synthesized the polymer and found in lecithin and monoolein black films the predicted behavior for an ion channel. Figure 2 illustrates the typical stepwise trend of current fluctuation across a membrane of monoolein doped with solutions of poly(DL-proline), where the positive and negative transitions of current course correspond to activation and deactivation of single ion channel formed by a polymer molecule.6 Recently, with the aim of elucidating the molecular mechanism of the ion conduction, we investigated the structures of two tetrameric derivatives, intermediates of the poly(DL-proline) synthesis, by X-ray crystal structure analysis, and we found that these structures in the solid state are characterized by the same regular sequence of the cis and trans conformations of the peptide bonds. Such a conformation appears, however, not suitable for interacting with ions, but it formally transforms into a spire of the channel conformation of poly(DL-proline) when the central peptide is changed by the cis to the trans con for ma ti or^.^-* The present paper investigates conformational changes in solutions of increasing ion concentrations, providing information on the active structures of pOly(DL-prOhe) during the ion passage across membranes. A suitable representation of the possible conformations of the tetrameric derivatives and the prediction of the most stable ion complexes structures are derived by theoretical energy calculations based on semiempirical van der Waals potential functions and electrostatic contributions.
The relative conformational rigidity and bulkiness of the proline residue freezes the torsional angle p about the N-C" bond and allows, when followed by another proline residue, only a very restricted range of the torsional angle $ about the Ca-C' bond. As a consequence, the structural changes in a polyproline chain are mainly due to transitions between the trans and cis conformations of the peptide bond except for the limited variance of the torsional angles ~pand $ as well as for the conformation of proline residue at the carboxyl end, which can assume also the &-helical local c o n f o r m a t i ~ n . ~ J ~ We have investigated the conformations allowed for BOC(DPro-L-Pro)20CH3 (Boc = tert-butoxycarbonyl) using the methods of theoretical conformational analysis and adopting our set of van der Waals potential functions and localized charges at the single atoms" and found that, except for one, all the 2s combinatorially possible cis-trans conformational sequences give rise to sterically allowed structures. The conformational parameters, van der Waals, and electrostatic energy contributions, evaluated after convergence of energy gradient minimization procedures, are reported in Table I. From the theoretical analysis, a large conformational degeneration emerges, which should suggest the presence in solution of several slowly (on the N M R scale) interconverting conformers. The presence of cations favors the conformer characterized by all-trans peptide conformations (TTTTDI2) because the positive
lp2
*To whom correspondence should be addressed at UniversitE! di Roma.
0022-3654/88/2092-4759$01.50/0
(1) Hladky, S. B.; Haydon, D. A. Biochim. Biophys. Acta 1972,274,294. (2) Ovchinnikov, Yu.A,; Ivanov, V. T.; Shkrob, A. M. Molecular Mech-
anism of Antibiotic Action on Protein Biosynthesis and Membranes; Elsevier
Scientific: Amsterdam, 1972. (3) De Santis, P.; Morosetti, S.; Rizzo, R. Macromolecules 1974, 7, 52. (4) Urry, D. W.; Goodall, M. C.; Glickson,J. D.; Mayers, D. F. Proc. Natl. Acad. Sci. U.S . A . 1971, 68, 1907. ( 5 ) Urry, D. W.; Venkatachalam, C. M.; Wood, S. A,; Prasad, K. U. Structure and Motion: Membranes, Nucleic Acids and Proteins; Adenine: New York, 1985. (6) De Santis, P.; Palleschi, A.; Savino, M.; Scipioni, A.; Sesta, B.; Verdini, A. Biophys. Chem. 1985, 21, 211. (7) Colapietro, M.; De Santis, P.; Nocilli, F.; Palleschi, A.; Spagna, R. Acta Crystallogr., Sect. C 1985, 41, 126. (8) Colapietro, M.; De Santis, P.; Palleschi, A.; Spagna, R. Biopolymers 1986, 25, 2227. (9) Ascoli, F.; De Santis, P.; Palleschi, A.; Rizzo, R. Peptides: Chemistry, Structure and Biology; Ann Arbor Science: Michigan, 1975. (10) De Santis, P.; Palleschi, A.; Savino, M.; Scipioni, A. Biophys. Chem. 1985. 21. 217. (1'1) Momany, F. H.; McGuire, R. F.; Burgess, A. W.; Scheraga, H. A. J . Phys. Chem. 1975, 79, 2361.
0 1988 American Chemical Society
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The Journal of Physical Chemistry, Vol. 92, No. 16, 1988
Figure 1.
De Santis et al.
-proline).
n
Ns'
100mV
60mV
K'
1
IPA
3.
Y
Figure 2. Current fluctuations of Na+ and K+ ions across a membrane of monoolein doped with poly(DL-proline).
charge in the center of the cavity not only eliminates the electrostatic repulsion between the C O groups but also provides an additional ion-dipole stabilizing factor.13-15 We have analyzed the structures of the possible ion complexes minimizing both the nonbonded and electrostatic intramolecular energies of the all-trans conformer as well as the ion-tetramer electrostatic energy.16 The deepest minimum corresponds to the cation inserted into a pseudooctahedral cavity of the tetramer giving rise to a 1/1 tetramer/cation complex with a fine structure depending on the ionic radius: the residual ion coordination site is occupied by a water molecule. The predicted structure of the Ba2+ complex is shown in Figure 3 as a stereoprojection. The other two relative energies minima correspond to the cation moved from the tetramer cavity in the opposite direction; this allows the tetramer to form partial coordinations with the ion. The remnant octahedral positions can be occupied by water molecules. These mixed complexes can be considered as intermediates of the dimeric 2/ 1 tetramer/ion complexes formation. Because of (12) The conformations are specified by the sequence of five letters (C and T indicate the peptide conformations cis and trans, respectively; A and D the two alternative conformations about the terminal ester group characterized by \Ir4 -53" and 147', respectively). (1 3) The conformation TTTTD provides the best complexing cavity because five CO groups realize a pseudooctahedral cage around the ion. (14) Ovchinnikov, Yu. A,; Ivanov, V. T. Tetrahedron 1974, 30, 1871. (15) Duax, W. L.; Smith, G . D. Biomolecular Stereodynamics; Adenine Press: New York, 1981. (16) In the refinement process, we considered the ionic charge centered on the ion and used the constraint xfonstant (d, - do)z, where d is the distance between the ion and t h e j t h oxygen of the CO groups and is the sum of the van der Waals radius of the oxygen atom and the ionic radius of the ion. In the calculations we have not considered the hydration molecules.
-
d,
Figure 3. Stereoview of the predicted most stable monomeric Ba2+
complex. the steric hindrance due to close contacts of the pyrrolidinic rings of the two tetramers, only the dimeric complex where the cation sees two different faces of the tetramer is allowed with an energy comparable to that of the monomeric complex. This is shown in Figure 4; a water molecule needs to complete the coordination sphere, as in the case of the monomeric complex. Thus only the two complexes TX and T2X (T = tetramer and X = cation) with close tetramer conformations should represent the state of the tetramer in solution in the presence of cations. Conformations of Boc(o-Pro-~-Pro)~OCH~ in solution were investigated by using NMR and CD spectroscopy.
Circular Dichroism Figure 5 shows CD spectra of the tetramer corresponding to the n-x* and x-x* electronic transitions of the peptide groups in different solvents. The spectra, in the considered solvents, show negative bands about 205-220 nm with different rotational strengths, surprisingly high for a molecule with an enantiomeric sequence of proline residues: the dissymmetric factor A€/€ is in It should be noted, however, that the range of (0.5-0.6) X only the conformational equivalence of the enantiomeric units results in a vanishing of the contributions to the optical activity except for the terminal effects. It is plausible that CD differences are the results of different proportions of the possible conformers in the various solvents. It is expected that the conformational sequence containing high proportions of cis-trans alternating peptide conformations, where the enantiomeric proline residues are conformationally different, mainly contribute to the CD bonds, whereas the all-trans and
Poly(DL-proline), an Ion Channel
The Journal of Physical Chemistry, Vol. 92, No. 16, 1988 4761
TABLE I: Conformational Parameters‘ of the Possible Conformers of the Free Oligopeptide Boc( D-Pro-L-Pro)20CH3 conformation codeb TTTTD TCTTD TTCTD TTTCD TCCTD TCTCD TTCCD TCCCD CTTTD CCTTD CTCTD CTTCD CCCTD CCTCD CTCCD CCCCD TTTTA TCTTA TTCTA TTTCA TCCTA TCTCA TTCCA TCCCA CTCTA CCTTA CTTCA CCCTA CCTCA CTCCA CCCCA
91
77 73 69 68 76 71 68 74 77 64 63 71 64 64 65 61 71 70 69 68 76 71 69 73 63 64 72 62 64 62 61
*I
-1 58 -119 -158 -152 -128 -131 -159 -1 30 -132 -130 -161 -161 -134 -132 -162 -138 -171 -135 -159 -153 -1 23 -130 -156 -122 -161 -137 -157 -138 -130 -159 -132
W1
92
179 2 179 177 3 1 178 3 -179 1 178 -179 2 1 179 2 180 0 179 178 3 2 177 3 178 -1 180 4 2 177 2
-78 -68 -74 -72 -62 -66 -76 -66 -69 -71 -75 -72 -63 -68 -75 -65 -77 -74 -75 -70 -65 -67 -78 -66 -77 -75 -71 -58 -67 -78 -66
$2
130 157 127 156 128 157 135 137 151 160 118 159 133 159 129 134 154 159 129 155 133 160 128 136 118 158 159 128 159 116 137
@2
93
$3
w3
9.,
$a
179 -179 -2 -176 -2 -178 -2 -3 -177 -179 -2 -178 -1 -177 -2 -2 179 180 -2 -178 -4 -179 -2 -2 -3 160 -179 -2 -178 -2 -2
65 70 67 81 69 74 64 62 76 71 68 78 68 75 65 65 66 66 65 73 58 71 64 64 64 66 77 71 71 66 65
-156 -154 -160 -121 -157 -126 -137 -139 -157 -156 -160 -1 30 -162 -126 -140 -145 -154 -147 -159 -138 -159 -142 -142 -148 -1 60 -147 -134 -163 -138 -148 -154
176 178 180 2 179 2 1 1 177 178 180 2 179 2 1 1 180 179 180 0 179 -1 0 -1 180 180 1 -176 -1 0 0
-76 -74 -6 9 -67 -68 -6 8 -6 8 -67 -76 -74 -68 -66 -67 -67 -68 -65 -64 -6 7 -72 -6 9 -74 -70 -66 -72 -7 1 -67 -68 -6 8 -73 -69 -6 9
135 143 145 150 146 156 145 151 120 144 147 154 148 160 148 156 -47 -47 -5 5 -55 -48 -60 -48 -60 -52 -46 -58 -44 -64 -53 -64
E, kcal/mol vdwC el“ totC -6.5 -6.5 -5.5 -5.7 -6.0 -5.2 -5.3 -6.9 -7.4 -7.7 -7.0 -7.5 -8.2 -6.5 -6.5 -8.2 -5.2 -5.8 -6.0 -5.5 -7.5 -5.3 -5.5 -7.0 -7.4 -7.2 -7.3 -8.9 -6.4 -7.0 -8.5
1.7 1.2 1.4 1.7 1.3 1.3 1.9 1.6 1.2 1.5 .9 1.7 1.5 1.4 1.4 2.1 1.7 1.1 1.7 1.9 1.2 1.2 1.8 1.2 1.2 1.3 1.5 1.8 1.3 1.3 1.8
-4.8 -5.3 -4.1 -4.0 -4.7 -3.9 -3.4 -5.3 -6.2 -6.2 -6.1 -5.8 -6.7 -5.1 -5.1 -6.1 -3.5 -4.7 -4.3 -3.6 -6.3 -4.1 -3.7 -5.8 -6.2 -5.9 -5.8 -7.1 -5.1 -5.7 -7.0
“IUPAC IUB conventions are followed for torsional angles 9, $, and w. *See footnote 12. Cvander Waals. “Electrostatic. ‘Total.
Figure 4. Stereoview of the predicted most stable dimeric Bat+ complex.
PO0
220
- I
24 0 1
-10
I
-20
Figure 5. CD spectra of 1 X lo4 M Boc(~-Pro-~-Pro)~oCH~ in water (-),
-
ethanol (-- -), and acetonitrile (- -).
all-cis conformations (where the monomeric units are conformationally equivalent) poorly contribute to the optical activity.
J
Figure 6. CD spectra of 1.0 X lo4 M Boc(~-Pro-~-Pro)~0CH, in acetonitrile at different r = Ba2+/T.
In fact, it is significant that increasing proportions of Ba(C10& and Ca(C10,)2 in acetonitrile solutions decrease the CD bands
De Santis et al.
The Journal of Physical Chemistry, Vol, 92, No. 16, 1988
4762
the monomeric units. In fact the spectrum in Figure 8 shows four equivalent quadruplets corresponding to the four different carbon atoms of the pyrrolidinic group; the relative chemical shifts suggest the trans conformation of the peptide groups. Figure 9 illustrates the changes of the proton spectrum by addition of increasing proportions of Ba(C10& between r (= Ba2+/tetramer) values of 0 and 0.5 The tetramer concentration was fixed at 1.0 X IO-* M. As can be seen, the original spectrum progressively disappears with the simultaneous emergence of new signals. The changes of the signals monitor the tetramer transconformations apparently in a single conformer Characterized by close values of CaH protons chemical shift; the four C"H protons correspond to four quadruplets (two of them almost coincident) characterized by similar constants (except at lower field). The ester values of J3C.HCeH) and Boc methyl original signals transform into two single peaks at lower field. A more accurate analysis reveals that the emergence of these signals is accompanied by minor signals, with similar chemical shift. This suggests the presence in solution of another conformationally similar complex with slow interconversion of the NMR scale. The formation of these two complexes follows different trends as monitored by the fractional area of the new and original methyl signals reported in Figure 9b. The formation of Ba2+ complexes is characterized by a simultaneous decrease of the original signals, which practically disappear about r = 0.7. This value also suggests the presence of two complexes characterized by r = 0.5 and r = 1, namely,
t
~
J
I
20-
Figure 7. CD spectra of 1.0 X lo4 M Boc(D-Pro-L-Pro)20CH3in acetonitrile at different r = Ca2+/T.
of the tetramer as shown in Figures 6 and 7, as should be expected if the cations induce transconformations (of the tetramer) to the all-trans ion complexing conformer.
NMR Spectra
-
The conformational changes of the proline residues are practically restricted to the cis trans peptide bond interconversion, whose relatively high energy barrier prevents the time averaging of NMR signals. As a consequence, in spite of the relatively low molecular weight, the tetraproline derivatives provide structural information about the single conformations present in solution. Figure 8 shows 13Cspectra of the tetramer in acetonitrile and after addition of Ba(C10&: the large multiplicity of signals, which derives from the conformational degeneration of the tetramer in solution, is dramatically reduced to the signals expected for a single structure characterized by a quasiconformational equivalence of
a
I
b
a
ll
C h
zn
40
30
Figure 8. ')C NMR spectra of 6.9 X IOv2 M Boc(D-Pro-L-Pro)20CH3(a) in acetonitrile and (b) after addition of Ba(C104)2.
10
Poly(DL-proline), an Ion Channel
The Journal of Physical Chemistry, Vol. 92, No. 16, 1988 4763
b C
M Boc(~-Pro-~-Pro),0CH,in CD$N at r (=Ba2'/T) 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 from bottom to Figure 9. Proton NMR spectra of 1.2 X top: (a) C'H region; (b) CH,(Boc) region; (c) CH,(ester) region.
5.0
I
I
4.0
4.0
,
4.4
I 1.1
,,-
I 4.0
Figure 11. Proton NMR, C"H region, of poly(DL-proline) in (a) CDCI,,
(b) CD3CN, (c) CD$N after addition of Ba(C104)2.
Figure 10. Proton NMR spectra of 1.1 X M Boc(D-PreLPro),OCH, in CD,CN, C'H region, at r (=Ca2'/T) 0.0, 0.1, 0.3, 0.5, 0.7, and 0.9 from bottom to top.
BaT, and BaT, which we can identify with the structures theoretically predicted. It is interesting that, while the original signals disappear, taking their chemical shifts constant, the arising signals move toward lower field as shown in Figure 9b. Spectral changes were observed also for the homologous calcium complexes as shown in Figure 10. In this case the exhaustion of the spectral changes occurs at about r = 0.8, suggesting the presence in solution of a larger proportion of the monomeric complex CaT.
The monomeric complex is, however, practically the only one observed in the case of Na+ and K+ complexes obtained in the same solvent at comparable tetramer c~ncentration.'~ N M R spectra in different solvents and in the presence of amounts of Ba2+were also obtained for poly(DL-proline). Figure 11 shows the proton spectrum of the CaH regions of the poly(DL-proline) in CDC13 and CD3CN; in the latter solvent, also, the spectrum obtained after addition of Ba(C10& is shown. The spectrum in chloroform solution is characterized by a strong splitting of C"H signals (1.1 ppm) with equal areas. This suggests the presence in solution of a dominant proportion of cis-trans alternating sequences, where the CaH protons are strongly differentiated in their chemical environments. Such a conformation corresponds to the crystal structure of the tetramer.8 The proportion of such a structure is reduced in acetonitrile solution, where the presence of a band at about 4.6 ppm suggests, according to the NMR results on the homologous tetramer, the presence of a consistent proportion of the all-trans structure. This is confirmed by the increasing and downfield shift of this band induced by (17) Submitted for publication in Biopolymers.
The Journal of Physical Chemistry, Vol. 92, No. 16, 1988
4764 I
Oh
De Santis et al.
I
1
X
b
5
0
5
10
r -
IC X
a .5
a 3 C I
-
Figure 12. (a) Molar fraction trends of the free tetramer cf),dimeric complex ( m ) , and monomeric and dimeric complexes ( m + d) versus r = Ba2+/T. The molar fractions are derived from the N M R peaks at 1.49 and 1.36 ppm cf). (b) Fitting of C D trend versus ppm ( m ) , 1.45 ppm (4, r = Ba2+/T as derived from the band at 205 nm.
adding of Ba2+. It is interesting to note the large width of the band as a consequence of a plausible increased rigidity of the all-trans channel structure when fitted by Ba2+ ions.
Discussion The results of the conformational calculations form the basis for a consistent interpretation of the CD and N M R data in terms of equilibria between the free tetramer, which exists in solution as a mixture of several conformations, and two ion complexes XT and XT2, characterized by similar all-trans conformations but with a different molecular complexity. It is straightforward to derive from the two equilibrium equations and the stoichiometric constraints the best pair of K, and Kd constants (m and d indicating the monomeric and dimeric complexes) capable of fitting with appropriate accuracy the trends of the C D bands as well as those of the N M R signals against the cation/tetramer ratio for different tetramer concentrations. The two constants can be written as K, = ( m / C f i ) ) T ' ; Kd = ( d / C f 2 i ) ) T 2
+
+ +
w i t h m + 2d f = 1 and r = ( m d i), wherem, d , and f are the monomeric and dimeric complexes and the free tetramer molar fractions, respectively, T is the total tetramer concentration, and finally r a n d i are the molar ratios of the total and of the free ions, respectively, and the tetramer. The equilibrium constant obtained were K , = 4.0 X lo3 M-' and Kd = 8.9 X lo5 M-2 in the case of Ba2+ and K, = 2.0 X lo5 M-' and Kd = 3.5 X lo7 M-2 in the case of Ca2+.
Figure 13. (a) Molar fraction trends of the monomeric ( m ) , dimeric (d) complexes, and the free tetramer cf) versus r = CaZ+/T. The molar fractions are derived from the N M R peaks at 1.50 ppm ( m ) , 1.45 ppm (d), and 1.37 ppm (b) Fitting of C D trend versus r = Ca2+/T as derived from the band at 200 nm.
m.
As an example, Figures 12 and 13 show the fitting of the NMR and CD data in the case of Ba2+and Ca2+ complexes; the NMR data refer to the changes of the Boc signals, which can be followed rather accurately. Notice that the molecular models obtained on the basis of the conformational calculations as well as the selfconsistence of the experimental data require the presence of a signal assignable to one of the two Boc methyl protons of the dimeric complex, under the larger signal at lower field (such a signal in fact shows a shoulder). N M R spectra of B o c ( ~ - P r o - ~ - P r o ) ~ 0 C H in, 10 times more dilute solution (tetramer concentration lod3M) in the presence of Ba(C10J2 show a different trend of the arising peaks assigned to the methyl groups of the monomeric and dimeric complexes, as expected according to the values of K, and Kd." Finally, the progressive downfield shift of all the signals assigned to the complexes for increasing proportions of cations indicates fast exchanges (on the N M R scale) of the complexed ions as supported also by relaxation time experiments carried out with 23Na.17 At present we are attempting to crystallize ion complexes of the tetraproline derivative to obtain the fine features of their structures. The hypothesis advanced in a previous paper6 that the poly(DL-proline) ability of activating the ion passage across membranes should be associated with the formation of channel conformations seems to be supported by the results obtained in this paper. The dimeric and monomeric complexes could represent, in fact, two steps of the ion pathway along the poly(DL-proline) channel. Registry No. B O C ( ~ - p r o - ~ - p r o ) ~ o C107500-78-7; H~, DL-proline homopolymer, 64298-80-2; pOly(DL-prOhe) SRU, 54325-06-3.
