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Long range electron transfer along an α-helix
286
Biochimica et Biophysica Acta, 1159(1992) 286-294 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34312
Long range electron transfer along an a-helix Hyosil Lee a, M. Faraggi b and Michael H. Klapper
a
Department of Chemistry, The Ohio State University, Columbus, OH (USA) and b Department of Chemistry, Nuclear Research Centre-Negec, Beer-Sheva (Israel)
(Received 24 March 1992)
Key words: Electron transfer; Long range electron transfer; Alpha helix; Pulse radiolysis;CD; Leucine zipper peptide The many observations of long range electron transfer in proteins raises the question of whether a protein's structure can influence the rate or path of such transfers, and if so, then how. To answer these questions requires information on which of the various structural elements composing proteins support long range electron transfer. In this report, we present evidence for long range electron transfer along the a-helix of a synthetic leucine zipper dimer. We also present electron transfer rate data obtained with other helical peptides.
Introduction Long Range Electron Transfer (LRET) in proteins, poeptides, and various other spacers over distances > 20 A is well-documented [1-8]. The questions that arise naturally are: (i), whether a protein's structure can affect the rate or the path of an L R E T process; and, thus, (ii), whether evolutionary pressures have led to structures that maximize the efficiency or control of L R E T in proteins. There is already suggestion of and evidence for the structural control of both electron transfer pathway and L R E T rate in proteins [9-11]. However, satisfactory answers to these questions require understanding the properties of L R E T within or across individual structural elements found in proteins. But whether this understanding can be acquired from the study of proteins alone is problematic. Protein structures are complex; there are the various structural details of the folded polypeptide chain and the many
Correspondence to: H. Lee, Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA. Abbreviations: Aib, a-methyl alanine; Dn, helical dimeric form of GCN4-6; drnaPhe, L-dimethylaminophenylalanine;e~q, the hydrated electron; GCN4-6, AcetyI-AVKQLEDKVWELLNIlNYKLENEVAKLKKLVAEA; LRET, long range electron transfer; OH, the hydroxyl radical; Mh, helical monomeric form of GCN4-6; Mrc, random monomeric form of GCN4-6; N3, the azide radical; pyrAla, L-pyrenylalanine; Trp', the neutral tryptophan indolyl radical; TyrO', the tyrosine phenoxy radical. While we have used the standard oneand three-letter abbreviations for the common amino acids, the following abbreviations have been introduced into reaction equations in order to describe more accurately the electron transfer between tyrosine and tryptophan: TrpH, tryptophan; TyrOH, tyrosine.
residues that might behave as electron transfer mediators. Because of this complexity, we have been studying L R E T in peptides. Our assumption has been that these potentially simpler models might be useful in isolating the various possible structural effects on the L R E T process. In this report, we present studies with peptides that form helical structures in solution. The L R E T system used in these studies is the pulse radiolyticaUy initiated electron transfer from tyrosine to the tryptophan radical ( T r p ) , an experimental system developed by Land and co-workers [12,13]. In the pulse-radiolysis experiment the hydrated electron (e~q) and the hydroxyl radical ( O H ) are primary radical species produced when high energy electrons (at about 3.5 MeV and in pulses of < 1 /~s from the linear accelerator in our laboratory) interact with the water of dilute aqueous solutions. In solutions with sufficient concentrations of both N 2 0 and azide, e~q and OH" are converted quantitatively to the azide radical (N 3) through the reactions: eaff +NeO+H + ~ N2 +OH"
(1)
O H + N 3 ~ N 3+OH
(2)
The aside radical rapidly and preferentially oxidizes the Trp-indole side-chain to the indolyl radical (Trp') in a peptide with one Tyr and one Trp separated by the spacer X. N.{+ TrpH-X-TyrOH---,Trp -X-TyrOH + Nj- + H +
(3)
T r p ; with a midpoint potential slightly greater than 1 V at pH 7 [14] can then oxidize the Tyr phenolic
287 side-chain to the phenoxy radical (TyrO') in the intramolecular reaction: Trp "-X-TyrOH ~ TrpH-X-TyrO"
(4)
Since both T y r O and T r p absorb strongly in the visible, it is easily shown that reaction 4 is a one-step process with stoichiometric electron transfer from the TyrOH donor to the Trp" acceptor [15]. But electron transfer can occur as either an intramolecular (unimolecular) or intermolecular (bimolecular) process, two pathways that can be distinguished kinetically. When the concentration of the free radical is much smaller than that of the peptide, then ket.app, the apparent electron transfer rate constant, is linearly dependent on the peptide concentration: ket,app = kun i + k bi(Trp-X-Tyr)
(5)
and the intramolecular rate constant is obtained from the intercept of the plot kaoo vs. peptide concentration. Our first goal in these studies has been to demonstrate unambiguously that LRET is possible within a helix. The peptides used in this study were based either on the 17 residue a-helix design of Marqusee and Baldwin [16] or the GCN4 coiled-coil design utilized by O'Shea and co-workers [17-19]. While parenthetic to this study, it is important for us to note that both TyrO" and Trp, the radicals with which we are dealing, are reported to have physiological significance [20-22]. Materials and Methods
We synthesized all peptides with the standard t-BOC protocol and a SAM II (Biosearch, Novato, CA) synthesizer. Synthesis began with the ultimate C-terminal amino acid attached to a MBHA (4-methylbenzhydrylamine) resin (BACHEM BioScience, Philadelphia, PA). The following was the synthetic cycle for the addition of each new residue. The t-BOC group on the growing peptide chain was removed with 45% trifluoroacetic acid (TFA) and 5% anisole (as a scavenger to minimize possible Trp oxidation) in dichloromethane (DCM). After neutralization with 10% diisopropylethylamine (DIPEA), the next t-BOC blocked amino-acid, added in approx. 6-fold excess, was coupled for approx. 2 h with 6.5% diisopropylcarbodiimide (DIPCI) in dimethylformamide (DMF). To minimize its acid destruction, the indole of tryptophan was protected by a formyl group. An amino-acid was coupled twice within one cycle in the case of bulky amino-acids, such as Val or Leu, or when the same amino-acid was linked consecutively. When Asn or Gln were added to the growing chain, 1.5 equivalents of 1-hydroxybenzotriazole hydrate (HOBt) was mixed with
the t-BOC amino-acid to minimize possible racemization. Unreacted growing chains with remaining free N-terminal amino groups were capped by reaction with 25% acetic anhydride in DCM. To add the next amino acid, the t-BOC group from the most recently added residue was removed by treatment with TFA and the synthesis cycle repeated. Upon completion of the chain, the final t-BOC group was removed and the N-terminal amino group acetylated with acetic anhydride. The resin bound peptide was then washed with methanol and stored at -20°C. To complete the synthesis, we removed the acid-labile side-chain protecting groups and cleaved the peptide from the resin with a trifluoromethanesulfonic acid/trifluoroacetic acid protocol [23]. Finally, we removed the formyl protecting group with a procedure supplied by Applied Biosystems [24]. Each peptide was purified first by passage over Sephadex G-10 equilibrated with 0.1 M ammonium acetate and then by HPLC with a semipreparative Ci8 reversed phase Dynamax Column (0.46 × 25 cm, 5 lz silica, 300 A pores, Rainin Instrument, CA) with acetonitrile/HzO gradients containing 0.1% trifluoroacetic acid. Peptide homogeneity was determined as a single peak in the HPLC. Peptide identity was verified by amino-acid composition and FAB-MS, both performed in The Ohio State University Campus Chemical Instrument Center. We also obtained UV spectra to verify that each peptide contained one Tyr and one Trp [25]. All CD measurements were taken with a JASCO J-500C spectropolarimeter (JASCO, Easton, MD) equipped with a water-jacketed 1-cm pathlength quartz cell maintained at +0.1°C over the range of 0-70°C. We determined the peptide concentration by measurement of the tyrosine plus tryptophan UV absorbance [25]. The CD instrument was calibrated with both ammonium-d-camphor-10-sulfonate [26] and o( - )pantolactone [27]. The pulse radiolysis procedures were those described elsewhere [15]. Rate constants were obtained by nonlinear fitting of absorbance-time profiles using the data acquisition package ASYST (Macmillan Software, New York, NY). All other nonlinear fits were done with either MINSQ (MicroMath Scientific Software, Salt Lake City, UT) or with a code developed in this laboratory from the GRIDLS subroutine of Bevington [28]. Using standard procedures, we redistilled all the solvents and reagents used in the peptide synthesis (DCM, DMF, DIPEA and DIPCI). The remaining chemicals were used as purchased: TFA (protein sequencing grade), anisole, acetic anhydride, HOBt and thioanisole from Aldrich (Milwaukee, WI); ethanolamine and trifluoromethanesulfonic acid from Sigma (St. Louis, MO). The CD standards were supplied by JASCO (ammonium-d-camphor-10-sulfonate) and Aldrich (o-(-)-pantolactone). All solutions were pre-
288 pared with water obtained from a Millipore Milli-Q apparatus.
.000
(a)
-.0Ol -.002
Results and Discussion L
LRET in leucine zipper peptide Our overall goal is to study the properties of the L R E T process in distinct structural elements of proteins. In this study, we sought definitive evidence that L R E T can occur in a helix. For this purpose we constructed a leucine zipper-type peptide. An apparently essential feature for the leucine zipper structure, a dimeric a-helical coiled coil, is an amino-acid sequence in which every seventh residue is a leucine (or related residue) with interspersed apolar residues forming a 4 - 3 apolar residue repeat [29,30]. When folded into an a-helix, these apolar 4 - 3 repeat residues align along the helix surface, thus, stabilizing a coiled-coil dimer with the 4 - 3 apolar residues of one chain in contact with those of the other. O'Shea and co-workers [17-19] have shown by both CD and I H - N M R measurements that a synthetic 33-residue peptide (GCN4-pl), taken from the yeast D N A binding transcriptional regulator GCN4, forms a stable parallel dimer of a-helices. Using G C N 4 - p l as a model we constructed a 33-met we call GCN4-6. Its structure is given below: AcetyI-AVKQLEDKVWELLNKNYKLENEVAKLKK-
-.003
"6 -.O04
X~
-,11115
-.006 270
I 280
I 290
I 300
I 310
i 320
I 330
I 340
Temp. (K) .000
(b) -.001 -.002
"~
-.11114
"O
-.005
-.006 270
280
I 300
290
I 310
I 320
I 330
I 340
Temp. (K)
Fig. l. Helix-coil transition with temperature for GCN4-6. Ellipticity was measured at 222 nm under the experimental conditions, 24/~M GCN4-6 (pH 7.0), 5 mM phosphate buffer, 1.0 M NaCI. (a), Data fit to equation generated from the three state equilibrium of Eqn. 7 in the text. Residuals are plotted in the inset; (b), data fit to equation generated from the two state-equilibrium of Eqn. 6 in the text.
LVAEA (The number 6 indicates that Tyr and Trp are separated by six intervening residues). In the design of GCN4-6 we replaced the following residues of GCN4pl: R1A, M2V, E10W, S14N, H18K, R25K, G31A, R33A. l Base on the proposed coiled-coil dimeric structure of G C N 4 - p l , the aromatic side-chains of the T y r O H / T r p " redox pair (in the 1-electron oxidized form of GCN4-6) should face into the aqueous environment with their fl-carbons separated by very nearly 10 A along a line parallel to the helix axis. We synthesized GCN4-6 using the methods described earlier. Its helical content, estimated from its ellipticity at 222 nm, is concentration-dependent at p H 7 (5 mM phosphate buffer), I°C and low salt ( < 0.2 M NaCI). At 1.0 M NaCI the helical content is independent of peptide concentration over the range 5 - 3 0 jzM and at I°C is roughly 70-80%. However, even at high salt, the apparent Tm for the helix-coil transition is dependent on the peptide concentration. These observations are consistent with a helical structure induced
i The changes introduced into the GCN4 peptide are indicated by a standard procedure, the one-letter abbreviation of the original amino acid followed by the residue position and the one-letter abbreviation of the new amino acid. For example, R1A indicates that Arg at position 1 has been replaced by an alanine.
by peptide aggregation. Assuming the simplest dimer model for the leucine zipper, we analyzed the melting curve of ellipticity vs. temperature on the basis of a two-state process in which the random coil m o n o m e r (Mrc) associates directly to the helical dimer ( D h ) . 2Mrc ~ Dh
(6)
The fit of the equation generated by this model to the observed data is adequate (Fig. lb), indicating that the random coil monomer in equilibrium with an ahelical dimer is a reasonable interpretation. However, there are small but unmistakable deviations between the data and the calculated best fit curve. We, therefore, considered the next more complex, possibility, a three-state model in which the m o n o m e r exists in both helical (M h) and random coil forms. 2Mrc ~ 2M h ~ D h
(7)
The three appropriate equations associated with this scheme, one mass balance and two equilibrium expressions, arc." PT = 2 ( D h ) + (Mh) + ( M r s ) Kh~I = ( M h ) / ( M r c ) = exp( - A H h d / R T + AShc I / R ) Kass = ( D h ) / ( M h ) 2 = exp( --
AH~/RT+
ASass / R )
(8)
289 PT is the total peptide concentration, ghe I the equilibrium constant for the m o n o m e r coil to helix transition and Kass the m o n o m e r helix to dimer association constant, The equilibrium constants have also been expressed in terms of their standard enthalpies and entropies. This set of nonlinear equations can be rearranged to quadratic expressions for the concentrations of the individual species. As an example, the concentration of the helical m o n o m e r is given by:
-.,
0~" '0
,
+ ~'~""~ + .."" - " ' ~... _'" ~ +" ~
10000
•
-
2P T
(9)
with similar quadratic expressions for D h and Mrc. The mean residue ellipticity is related to the concentrations of the three species by: O = [O]M~(Mrc) +2[O]oh(Dh) + [O]M.(Mh)
40
o...monomer 1000
0
.' "
"'"'--.
60© 3.00
'
20
0
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~
:1.14
*.28
' 1.4t
~ 3.56
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0 $.70
1/T (K'lxl0))
ASas s / R )
exp(AHass / R T - ASass/R)= 0,
00
(2-.
.~
2(Mh) 2 + (Mh)[1 + exp(A Hh~I / R T - SheI/R)]
× exp(AHa,s/RT -
100
(]o)
where the [O]Mrc, [O]Mh, [O]Dh are the molar mean residue ellipticities of the random coil monomer, the helical m o n o m e r and the dimer, respectively. Assuming that [O]Dh=[O]Mh, we fit Eqn. 10 plus the three quadratic expressions (9) to the experimental data. In this nonlinear regression analysis based on the G R I D L algorithm of Bevington [28], the constants left free to vary were [O]M,, [O]M,c, AHhet, AShel, AHass and ASas s. As seen in Fig. la, this model yields a reasonable fit. We, therefore, suggest that while the simpler d i m e r / m o n o m e r model of Eqn. 6 describes the essential features of the h e l i x / r a n d o m coil transition in CGN4-6, an additional perturbation must be included in the final scheme. Whether this correction is sufficient for the complete determination of the aggregation process must await higher precision data. Thus, formally, we still have only an incomplete understanding of the GCN4-6 melting behavior. Nevertheless, the CD data clearly indicate that: (i), in solution near 0°C and at sufficiently high concentrations GCN4-6 occurs predominantly as an aggregate, most likely as a dimer, in which; (ii), each chain has a helical content of 7 0 - 8 0 % (The fit to the model of Eqn. 7 also suggests that those chains which are monomeric at this low temperature will be predominantly helical.) and (iii), at the higher temperatures investigated the chains exist as random coils. Finally, the diffraction pattern obtained from crystals of GCN4-6 is consistent with the coiled-coil parallel dimer structure of the leucine zipper reported for G C N 4 - p l (communication from M. Sundaralingam). We then looked for the L R E T process of Eqn. 4 in the GCN4-6 helical dimer, i.e., the reduction of T r p ' by a tyrosine side-chain when these redox partners are held apart by an a-helix. Our approach was to assume that the L R E T rate in a helical structure would not be
Fig. 2. Temperature dependence of electron transfer in GCN4-6, (o), GCN4-6: measured electron transfer rates under experimental conditions as in Fig. 1, except for the addition of 0.01 M N3 . (+), 17(Aib)-6, experimental conditions are those of Fig. 6. (. . . . . . ), monomer (%) calculated from constants obtained in the fit to the three-state model of Fig. 1. ( . . . . . . ), dimer (%) calculated from constants obtained in the fit to the three-state model of Fig. 1. identical to the electron transfer rate in a nonhelical structure. Thus, the t e m p e r a t u r e dependence of the L R E T process would reflect the electron transfer activation energies in both structures as well as the helixcoil transition enthalpy. Were this the case, then the Arrhenius plot (In kapo vs. 1/T) would be nonlinear over the t e m p e r a t u r e range of the helix-coil transition. To measure the electron transfer of reaction (4) in GCN4-6, we used the pulse radiolysis procedures described elsewhere [15]. The electron transfers we did observe were intra-, not intermolecular, since the bimolecular process makes a negligible contribution at the low peptide concentrations employed in these experiments [15]. As seen in Fig. 2, the Arrhenius plot (In kapp vs. 1/T) obtained with GCN4-6 is not linear. Moreover, there is an obvious transition in the electron transfer process, a transition that coincides with helixto-coil melting. Since GCN4-6 is helical at low temperatures (see above), we shall conclude that electron transfer in GCN4-6 at low temperature occurs across the helix structure and, therefore, must be a long range process. However, before reaching this conclusion we must consider another possible scheme. Were electron transfer only possible in the random coil and not the a-helical structure, then the observed electron transfer at low temperatures would be due to that small amount of random coil in equilibrium with the a-helical structure. Trp '-(helix)-TyrOH------//---,TrpH-(helix)-TyrO" ][fast
Trp -(coil)-TyrOH
fast
l[fast
(11)
, TrpH-(coil)-TyrO
This possibility derives from the observations that the peptide is not completely helical at low temperatures, as measured by CD and that linear extrapolation from
290 the high-temperature data to the low:temperature region would suggest an electron transfer in the random coil almost one order of magnitude faster than the electron transfer observed in the putative a-helical dimer. However, the reaction scheme of Eqn. 11 is not consistent with the data. Were the nonreactive helical structure in slow (relative to the rate of electron transfer) equilibrium with the reactive random coil, or were the conversion of helix-to-coil rate determining, then in the temperature region of the transition, where both random coil monomer and helical dimer must be present, we should observe a biexponential process. The faster phase would be from those random monomeric chains in the equilibrium established before the start of the reaction; the slower phase would arise from the slower obligatory conversion of the non-reactive a-helix to the reactive random coil. Additionally, the relative magnitudes of the two phases should be temperature dependent, the slower phase accounting for most of the reaction at low-temperatures and the faster accounting for most of the reaction at the high. At no temperature did we observe two phases of Trp" reduction. Fig. 3 presents, as one example, the results of a kinetic experiment at 24°C, a temperature at which there is approx. 50% helical structure. This example shows the initial oxidation of Trp by N 3 as seen by the fast rise in absorbance at 510 nm due to the formation of T r p - The subsequent slower decay of Trp" back to Trp is due to electron transfer from the electron donor, the tyrosine side-chain. (The absorbance at 510 nm does not decay back to zero because of the finite equilibrium between the tyrosine and tryptophan radicals [15].) The experimental points have been fit by least squares to a biexponential equation; the first phase representing Trp oxidation, the second Trp" reduction. The good fit indicates that there is only one exponential process associated with the reduction of Trp" by tyrosine.
5.50
4,30
0
3.10
o
1.90
0.70
-0.50 0.00
020
0.40
0.60
0,80
1 O0
Time (msec) Fig. 3. Electron transfer in GCN4-6. Absorbance measured at 510 nm as a function of time under experimental conditions identical to those for the GCN4-6 data of Fig. 2. (o), Experimental points; ( ), best-fit calculated time-dependence based on the equation A = a 0 + a 1 e x p ( - k i t ) + a 2 e x p ( - k2t)
Therefore, for the scheme of Eqn. 11 to apply chain unfolding would have to be fast relative to electron transfer in the random coil. In this case, were the chain unfolding enthalpy comparable to or less than the activation energy of electron transfer in the random structured chain, then we would not expect the large nonlinearity observed (see below). On the other hand, were the unfolding enthalpy higher than the activation energy of electron transfer in the random chain, then the low-temperature region of the Arrhenius plot should be either linear (not concave up) or in a more complex (e.g., different steps in the unfolding process being rate determining at different temperatures) situation concave down. That the low-temperature data points turn upwards is evidence for a low-temperature form in which electron transfer is slower than that expected in the random coil existing at the same temperature, with both structures in rapid equilibrium. Since the predominating structure at low-temperature is the a-helical structure, this low rate constant must reflect electron transfer in the dimer. There is one last consideration. The CD data suggest that the chains within the dimer are not completely helical. The most likely interpretation is chain unravelling at the two ends. However, the redox d o n o r / a c c e p t o r pair is located well within the center of the helical structure, so that one cannot postulate direct contact between donor and acceptor due to chain flexibility at the two ends. Therefore, since there is electron transfer in GCN4-6 dimer at low temperature and, since T r p and Tyr are separated by a helical structure that does not permit contact between them, electron transfer must occur across the helix structure and must be a long range process. The estimated linear extrapolation from higher to lower temperatures suggests that the L R E T rate in the leucine zipper is almost one order of magnitude slower than the electron transfer rate in the random coil at the same low temperature. How is this explained? It is possible that the helical structure of GCN4-6 slows electron transfer because of some structural or geometric feature yet to be explained. However, electron transfer in the random coil could occur by direct contact between the donor and acceptor residues, a potentially faster process than LRET. Were this the predominant path of electron transfer in the random coil, then the mechanism of electron transfer would be different in the two structures, contact in the random structure and L R E T in the helical. At present, therefore, we cannot directly compare electron transfer rates in the two structures of GCN4-6. We can, however, note interesting comparisons in related peptides. Thus, at I°C, a temperature at which GCN4-6 is helical, the L R E T rate between Tyr and Trp" separated by an extended - p r o 3- chain is approx. 10-fold slower [31] than the rate across the a-helical GCN4-6 spacer. The
291 o
through space distance in both cases is approx. 10 A, but the through backbone distance in GCN4-6 is roughly twice that in Trp-(pro)3-Tyr. On the other hand, at 25°C the observed rate constants for LRET between TyrOH and Trp" separated by oligoglycine (12,15), oligoglutamate (15) and the 17(Aib)-n peptides (see below) with only a few exceptions fall into the range of (1-5). 104 s -1 and show only a negligible dependence on chain length. This is in contrast to the results we have obtained with the proline spacer (37).
(a)
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E ~
10-
% 0 ID "~
5-
0
(b)
% X
i
i
i
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~
i
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Electron transfer in the peptide 17(Aib)-n We have also looked for electron transfer in a series of peptides based on the 17-residue design of Marqusee and Baldwin [16]. The a-helices of these peptides may be stabilized by glutamate and lysine i + 4 ion-pair interactions and by a favorable interaction between the charges on the chain and the helix dipole. In the peptides we synthesized, we varied the number of residues between the Trp and Tyr, with Tyr located at the C-terminal end of the chain. A typical peptide with 7 intervening residues is: AcetyI-AEUAAKEUAWAKEUAAKY-NH 2 U is the one-letter abbreviation for a-methyl alanine (2-amino isobutyric acid) also abbreviated as Aib. For convenience we have named this illustrated peptide 17(Aib)-7; the first number refers to the number of residues in the peptide and the second to the number of residues between the Trp and Tyr. Because of steric interactions, the ~b,y angles associated with the Aib residue are constrained to a region of the Ramachandran map that corresponds to the a-helix or the 310helix and small Aib containing peptides contain either of these structures (see Refs. 32,33 and references therein). We, therefore, included Aib residues in an attempt to stabilize further the helical content of the peptides synthesized for this study. Our peptides had 3, 6, 7, 8, 11 and 13 intervening residues. In each case Trp was substituted for either Ala (n = 6, 7, 11) or Aib (n = 3, 8, 13). All the synthetic 17(Alb)-n peptides were at least partially a-helical at I°C, as seen from the characteristic CD spectrum with troughs located at 208 and 222 nm. We estimated the helicity of each from the 222 trough by assuming 3000 deg cm z dmo1-1 for the molar residue ellipticity of the random structure and calculating an a-helical content with the procedure of Chen et al. [34]. Each peptide, except 17(Aib)-3 with a greater apparent helicity, has an apparent helical content near 60% (range, 57-64%; average, 60.2 + 2.3%) at 1°C, F < 0.1, pH 7.0. (Since the ellipticity does not reach a constant value as the temperature approaches 0°C, we cannot determine whether 60% of all the peptide chains are completely helical, with the remain-
i
S-
~
1'0
1'~
pH Fig. 4. Helical content of monomeric peotides as a function of pH. Ellipticity at 222 nm measured at I°C in a solution containing 1 mM sodium citrate, 1 mM sodium borate, 1 mM sodium phosphate dibasic and 0.01 M NaCl; pH adjusted with NaOH or HCI. Top, 17(Ala)-6; bottom, 17(Aib)-7.
ing 40% in a coil form, or whether all of the chains contain both structures and, thus, are 60% helical and 40% coil.) This helical content is independent of concentration over a range of 8-30 I~M. While the helicities of the peptides synthesized in Baldwin's laboratory are higher, those of the 17(Aib)-n peptides may be consistent with the reported helix destabilization by Trp [35]. The measured ellipticities, of all the 17(Aib)-n peptides: (i), depend on pH (Fig. 4) - constant between pH 2 and 8 and decrease at higher pH); (ii), decrease with increasing NaCI concentration; and (iii), decrease with increasing temperature. In analyzing helix temperature dependencies we assumed a noncooperative two-state model, with a simple equilibrium between helical and nonhelical structures. Consistent with this model, we obtained linear van't Hoff plots in each case. The estimated unfolding enthalpies were approx. 16 kJ/mol (range 14-20 kJ/mol; avg. 16 +_ 0.3 kJ/mol). These enthalpies are consistent with those reported by Merutka and Stellwagen [35], who worked with closely related peptides, but are a good deal lower than the approx. 42 kJ/mol reported later from this same laboratory [36]. We should note that while consistent with the simple two state melting model used in the fitting, the collected data cannot rule out a cooperative transition. Thus, the computed values of the unfolding enthalpies would be sensitive to the validity of the model on which the fitting was based. Our reported enthalpies are only preliminary estimates. While we included Aib in order to stabilize the a-helical structure of the synthesized peptides, there appears to be little stabilization at neutral pH. 17(Aib)6 and 17(Ala)-6, in which the three Aib residues were
292 replaced with Ala, had the same a-helical contents, 61% and 62%, respectively. Moreover, the unfolding enthalpy, 20 kJ/mol, of 17(Ala)-6, is within experimental error, that of the average for all the Aib peptides. However, while the Aib peptides maintain a constant helicity as the pH is lowered below 7, the helicity of 17(Ala)-6 decreases at lower pH (Fig. 4). Thus, at acidic pH, Aib does appear to stabilize a helical structure. While we cannot yet explain this apparent pH dependence of Aib helix stabilization, these results suggest that the Aib contribution to the stabilization of helices is not a simple linear (i.e., additive) property. With each of the synthesized 17(Aib)-n we measured electron transfer rates as a function of temperature. As in the GCN4-6 case, we expected the Arrhenius plots for the measured first-order intramolecular rate constants to be nonlinear. Once again the low concentrations of the peptides insured against a significant bimolecular reaction. At approx. 1°C, pH 7, in 5 mM phosphate buffer plus 0.01 M NaN 3 (added for the initiation of the electron transfer) the apparent first order electron transfer rate constant in the 17(Aib)-n peptide series increases from 1.9- 103 s- i in 17(Aib)-13, to 17 • 103 s - t in 17(Aib)-3. The observed rate constant dependence on amino-acid separation (Fig. 5) appears to be exponential, as would be predicted by Marcus theory for an L R E T process [37]. For comparison, Fig. 5 also contains earlier distance dependence data for T y r O H / T r p electron-transfer across the oligoproline spacer [38]. The inflexibility of the proline residue insures that the T y r O H and Trp" residues cannot come into contact and electron transfer must be long range. With the oligoproline spacer L R E T is relatively insensitive to the separation distance; i.e.,/3ap o, the slope of the In kap o v s . distance curve, may be as low as 0.27 ,~-l. Were electron transfer in 17(Aib)-n an L R E T process, then the apparent distance, in terms of the peptide main chain, dependence of the rate constant would have to be even shallower.
{:3 10000
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100
I
L
2
&
I
i
4
number
i
i
6
; 8
of
i
I
10
i
~
12
i
14
residues
Fig. 5. Electron transfer rate constant dependence on 17(Aib)-n chain length. (13 13), 17(Aib)-n peptides at approx. I°C, pH 7, 5 m M phosphate buffer, 0.01 M NAN3; ( zx zx), Tyr-(Pro)n-Tr p at 25°C. Data taken from Ref. 38.
100000
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10000
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3000 2.80
I
i
i
I
~.oo
3.=o
3.40
3.80
lIT
(K"xI0
3.80
~)
Fig. 6. Temperature-dependence of electron transfer rate constant with 17(Aib)-7. Approx. 0-60°C, pH 7, 5 mM phosphate buffer, 0.01 M NaN 3.
We also determined the temperature dependence of the electron transfer rate constant with each of the 17(Aib)n peptides. Over the range of approx. 0-60°C the helix content of each peptide decreases from approx. 60% to approx. 10% as measured by CD. Unlike the GCN4-6 experiment, the Arrhenius plots were all linear over this temperature range where the peptide helices unravel. Example data for 17(Aib)-7 are in Fig. 6 and for 17(Aib)-6 in Fig. 2. The estimated activation energies obtained from all these linear Arrhenius plots are near 23 k J / m o l (range 21-25 kJ/mol; avg. 23 + 1.7 kJ/mol). This is similar to the L R E T E a of approx. 21 k J / m o l found when this same amino-acid pair is separated by the oligoproline spacer [38]. Moreover, the electron activation energies found with Trp-Tyr, 21 k J / m o l [12]; Tyr-Trp, 22 k J / m o l [38] and Tyr-Glu-GluTrp, 21 k J / m o l [16], are also closely similar. While one could argue that there is L R E T across an a-helical structure at the lower temperatures even though the Arrhenius plots obtained with the 17(Aib)-n peptides are linear, there is another interpretation for the observed linearity. Were electron transfer sufficiently slow in the a-helix and were the equilibrium between helix and coil sufficiently fast, then electron transfer need not occur in the helical structure at all. This explanation would be represented by a scheme similar to that of Eqn. 11. There is initial fast unfolding followed by electron transfer in the nonhelical form, but there could also be a slower L R E T in the helical form. In the GCN4-6 case, we ruled out an initial unfolding scheme by appealing to kinetic arguments and to the shape of the nonlinear Arrhenius plot. On the other hand, the unfolding enthalpies obtained from the CD dependencies of the 17(Aib)-n peptides are less than 16 + 0.3 k J / m o l , though close to the E a we measured for the electron transfers. Hence, we would be reluctant to conclude that we have observed an L R E T process in the 17(Aib)-n peptides. Comparing the GCN4-6 and 17(Aib)-6 data (Fig. 2) allows further interpretation of the 17(Aib)-n data.
293 Above 25°C, the rate constants for electron transfer in GCN4-6 coincide with the rate constants for electron transfer in 17(Aib)-6. The two peptides are analogous in that 6 residues separate Trp" and T y r O H in both. Because the CD results suggest that both 17(Aib)-6 and GCN4-6 are largely random monomers at these higher temperatures, we argue that the coincidence of the electron transfer rates is not coincidental. Rather, the rate constants in both peptides at these higher temperatures must be generic and associated with electron transfer in the nonhelical structure. Below 25°C, the rate constants obtained with GCN4-6 are clearly lower than those obtained with 17(Aib)-6. The linearity of the 17(Aib)-6 results together with the difference of rates between 17(Aib)-6 and GCN4-6 at low-temperatures supports the proposed initial unfolding reaction scheme. We conclude that electron transfer in the 17(Aib)-n series of peptides at all measured temperatures occurs primarily through the nonhelical structure. However, whether electron transfer in the random structure occurs by direct contact between electron donor and acceptor or is long-range hinges on whether the rate of d o n o r / a c c e p t o r 'collision' is rapid or slow relative to the observed electron transfer. With respect to this question, there are at least two observations to be considered. First, previous calculations [39,40] have shown that the &,~ dihedral angles permitted for the Aib residue occur within two restricted regions on the Ramachandran plot - near - 5 7 °, - 4 7 ° and +57 °, +47 °. In these two regions are found right and left handed a-(or 310-)helices respectively. This explains why the Aib residue should help to stabilize a helical structure. However, this steric restriction on rotation about the N-C ~ and the C'~-C backbone bonds must make peptides that contain Aib less flexible than those that do not. There is one Aib residue between Tyr and Trp in 17(Aib)-6; there are no Aib residues in GCN4-6. Yet the rates of electron transfer are closely similar in the two 'random' coils. Second, electron transfer in the 17(Aib)-n series is exponentially dependent on the number of amino-acid residues in the chain between donor and acceptor. Is this dependence, which is consistent with an L R E T mechanism, also consistent with a contact electron transfer mechanism? In conclusion, we have argued for an L R E T process in the a-helical structure of the GCN4-6 dimer. Moreover, while the rate of electron transfer appears to be slower in the helix than in the random coil, we cannot directly compare the two, for we do not know whether electron transfer in the random structure is a contact or long range process. Other laboratories have also reported electron transfer across helical structures [41-43]. However, there was no careful consideration in these reports of whether the local structures over which electron transfer occurred were indeed helical. Our results suggest that conclusions of L R E T across
helices must be considered carefully, since electron transfer could occur after rapid melting to an unstructured form of the peptide.
Acknowledgements We are indebted to Edward Ray who has maintained our linear accelerator permitting the pulse radiolysis experiments described. This work was supported by grant GM 35718 from the National institutes of Health.
References 1 Klapper, M.H. and Faraggi, M. (1979) Q. Rev. Biophys. 12, 465-519. 2 Isied, S.S. (1984) Prog. Inorg. Chem. 32, 443-517. 3 Guarr, T. and McLendon, G. (1985) Coord. Chem. Rev. 68, 1-52. 4 Hush, N.S. (1985) Coord. Chem. Rev. 64, 135-157. 5 Peterson-Kennedy, S.E., McGourty, J.L., Ho, P.S., Sutoris, C.J., Liang, N., Zemel, H., Blough, N.V., Margoliash, E. and Hoffman, B.M. (1985) Coord. Chem. Rev. 64, 125-133. 6 Mayo, S.L., Ellis, W.R., Crutchley R.J. and Gray, H.B. (1986) Science 233, 948-952. 7 Miller, J.R. (1987) Nouv. J. Chim. 11, 83-89. 8 McLendon, G. (1988) Acc. Chem. Research 21, 160-167. 9 Michel-Beyerle, M.E., Plato, M., Deisenhofer, J., Michel, H., Bixon, M. and Jortner, J. (1988) Biochim. Biophys. Acta 932, 52-70. 10 Gray, H.B. and Malmstrom, B.G. (1989) Biochemistry 28, 74997505. 11 Weinstein, M., Alfassi, Z.B., DeFelippis, M.R., Klapper, M.H. and Faraggi, M. (1991) Biochim. Biophys. Acta 1076, 173-178. 12 Priitz, W.A., Land E.J. and Sloper, R.W. (1981) J. Chem. Soc. Faraday Trans I. 77, 281-292. 13 Butler, J., Land, E.J., Priitz, W.A. and Swallow, A.J. (1982) Biochem. Biophys. Acta 705, 150-162. 14 DeFelippis, M.R., Murthy, C.P., Faraggi, M. and Klapper, M.H. (1989) Biochemistry 28, 4847-4853. 15 Faraggi, M., DeFelippis, M.R. and Klapper, M.H. (1989) J. Am. Chem. Soc. 111, 5141-5145. 16 Marqusee, S. and Baldwin, R.L. (1987) Proc. Natl. Acad. Sci. USA 84, 8898-8902. 17 O'Shea, E.K., Rutkowski, R. and Kim, P.S. (1989) Science 243, 538-542. 18 O'Shea, E.K., Rutkowski, R., Stafford W.F. and Kim, P.S. (1989) Science 245, 646-648. 19 Oas, T.G., McIntosh, L.P., O'Shea, E.K., Dahlquist, F.W. and Kim, P.S. (1990) Biochemistry 29, 2891-2894. 20 Barry, B.A. and Babcock, G.T. (1987) Proc. Natl. Acad. Sci. USA 84, 7099-7103. 21 Stubbe, J.A (1989) Annu. Rev. Biochem. 58, 257-285. 22 Sivaraja, M., Goodin, D.B., Smith M. and Hoffman, B.M. (1989) Science 245, 738-740. 23 Bergot, B.J., Noble, R.F. and Geijer, T. (1987) Proc. Eur. Pept. Sym. 97-101. 24 Peptide Synthesizer, User Bulletin, No. 17 (Applied Biosystems, Foster City, CA, 1987). 25 Edelhoch, H. (1967) Biochemistry 6, 1948-1954. 26 Takakuwa, T., Konno, T. and Meguro, H. (1985) Anal. Sci. 1, 215-218. 27 Konno, T., Meguro, H. and Tuzimura, K. (1975) Anal. Biochem. 67, 226-232.
294 28 Bevington, P.R. (1969) "Data Reduction and Error Analysis for the Physical Sciences", McGraw-Hill, New York. 29 Landschulz, W.H., Johnson, P.F. and McKnight, S.L. (1988) Science 240, 1759-1764. 30 Landschulz, W.H., Johnson, P.F. and McKnight, S.L. (1989) Science 243, 1681-1688. 31 DeFelippis, M.R. (1990) 'The Redox Potentials of the Tyrosine and Tryptophan Radicals and Long-Range Electron Transfer Between tyrosine and Tryptophan in Peptides', Ph.D. thesis. 32 Nagaraj, R. and Balaram, P. (1981) Acc. Chem. Res. 14, 356-362. 33 Karle, E.L. and Balaram, P. (1990) Biochemistry 29, 6747-6756. 34 Chen, Y.-H., Yang, J.T. and Chau, K.H. (1974) Biochemistry 13, 3350-3359. 35 Merutka, G., Lipton, W., Shalongo, W., Park, S.-H. and Stellwagen, E. (1990) Biochemistry 29, 7511-7515.
36 Merutka, G. and Stellwagen, E. (1990) Biochemistry 29, 894-898. 37 Marcus, R.A. and Sutin, N. (1985) Biochim. Biophys. Acta 811, 265-322. 38 DeFelippis, M.R., Faraggi, M. and Klapper, M.H. (1990) J. Am. Chem. Soc. 112, 5640-5642. 39 Marshall, G.R. and Bosshard, H.E. (1972) Circ. Res. 30/31 (Supp II), 143-150. 40 Burgess, A.W. and Leach, S.J. (1973) Biopolymers, 12, 2599-2605. 41 Inai, Y., Sisido, M. and Imanishi, Y. (1991) J. Phys. Chem. 95, 3847-3851. 42 Tamiaki, H. and Maruyama, K. (1991) J. Chem. Soc. Perkins 1, 817-822. 43 Basu, G., Kubasik, M., Anglos, D., Secor, B. and Kuki, A. (1990) J. Am. Chem. Soc. 112, 9410-9411.
Biochimica et Biophysica Acta, 1159(1992) 286-294 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34312
Long range electron transfer along an a-helix Hyosil Lee a, M. Faraggi b and Michael H. Klapper
a
Department of Chemistry, The Ohio State University, Columbus, OH (USA) and b Department of Chemistry, Nuclear Research Centre-Negec, Beer-Sheva (Israel)
(Received 24 March 1992)
Key words: Electron transfer; Long range electron transfer; Alpha helix; Pulse radiolysis;CD; Leucine zipper peptide The many observations of long range electron transfer in proteins raises the question of whether a protein's structure can influence the rate or path of such transfers, and if so, then how. To answer these questions requires information on which of the various structural elements composing proteins support long range electron transfer. In this report, we present evidence for long range electron transfer along the a-helix of a synthetic leucine zipper dimer. We also present electron transfer rate data obtained with other helical peptides.
Introduction Long Range Electron Transfer (LRET) in proteins, poeptides, and various other spacers over distances > 20 A is well-documented [1-8]. The questions that arise naturally are: (i), whether a protein's structure can affect the rate or the path of an L R E T process; and, thus, (ii), whether evolutionary pressures have led to structures that maximize the efficiency or control of L R E T in proteins. There is already suggestion of and evidence for the structural control of both electron transfer pathway and L R E T rate in proteins [9-11]. However, satisfactory answers to these questions require understanding the properties of L R E T within or across individual structural elements found in proteins. But whether this understanding can be acquired from the study of proteins alone is problematic. Protein structures are complex; there are the various structural details of the folded polypeptide chain and the many
Correspondence to: H. Lee, Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA. Abbreviations: Aib, a-methyl alanine; Dn, helical dimeric form of GCN4-6; drnaPhe, L-dimethylaminophenylalanine;e~q, the hydrated electron; GCN4-6, AcetyI-AVKQLEDKVWELLNIlNYKLENEVAKLKKLVAEA; LRET, long range electron transfer; OH, the hydroxyl radical; Mh, helical monomeric form of GCN4-6; Mrc, random monomeric form of GCN4-6; N3, the azide radical; pyrAla, L-pyrenylalanine; Trp', the neutral tryptophan indolyl radical; TyrO', the tyrosine phenoxy radical. While we have used the standard oneand three-letter abbreviations for the common amino acids, the following abbreviations have been introduced into reaction equations in order to describe more accurately the electron transfer between tyrosine and tryptophan: TrpH, tryptophan; TyrOH, tyrosine.
residues that might behave as electron transfer mediators. Because of this complexity, we have been studying L R E T in peptides. Our assumption has been that these potentially simpler models might be useful in isolating the various possible structural effects on the L R E T process. In this report, we present studies with peptides that form helical structures in solution. The L R E T system used in these studies is the pulse radiolyticaUy initiated electron transfer from tyrosine to the tryptophan radical ( T r p ) , an experimental system developed by Land and co-workers [12,13]. In the pulse-radiolysis experiment the hydrated electron (e~q) and the hydroxyl radical ( O H ) are primary radical species produced when high energy electrons (at about 3.5 MeV and in pulses of < 1 /~s from the linear accelerator in our laboratory) interact with the water of dilute aqueous solutions. In solutions with sufficient concentrations of both N 2 0 and azide, e~q and OH" are converted quantitatively to the azide radical (N 3) through the reactions: eaff +NeO+H + ~ N2 +OH"
(1)
O H + N 3 ~ N 3+OH
(2)
The aside radical rapidly and preferentially oxidizes the Trp-indole side-chain to the indolyl radical (Trp') in a peptide with one Tyr and one Trp separated by the spacer X. N.{+ TrpH-X-TyrOH---,Trp -X-TyrOH + Nj- + H +
(3)
T r p ; with a midpoint potential slightly greater than 1 V at pH 7 [14] can then oxidize the Tyr phenolic
287 side-chain to the phenoxy radical (TyrO') in the intramolecular reaction: Trp "-X-TyrOH ~ TrpH-X-TyrO"
(4)
Since both T y r O and T r p absorb strongly in the visible, it is easily shown that reaction 4 is a one-step process with stoichiometric electron transfer from the TyrOH donor to the Trp" acceptor [15]. But electron transfer can occur as either an intramolecular (unimolecular) or intermolecular (bimolecular) process, two pathways that can be distinguished kinetically. When the concentration of the free radical is much smaller than that of the peptide, then ket.app, the apparent electron transfer rate constant, is linearly dependent on the peptide concentration: ket,app = kun i + k bi(Trp-X-Tyr)
(5)
and the intramolecular rate constant is obtained from the intercept of the plot kaoo vs. peptide concentration. Our first goal in these studies has been to demonstrate unambiguously that LRET is possible within a helix. The peptides used in this study were based either on the 17 residue a-helix design of Marqusee and Baldwin [16] or the GCN4 coiled-coil design utilized by O'Shea and co-workers [17-19]. While parenthetic to this study, it is important for us to note that both TyrO" and Trp, the radicals with which we are dealing, are reported to have physiological significance [20-22]. Materials and Methods
We synthesized all peptides with the standard t-BOC protocol and a SAM II (Biosearch, Novato, CA) synthesizer. Synthesis began with the ultimate C-terminal amino acid attached to a MBHA (4-methylbenzhydrylamine) resin (BACHEM BioScience, Philadelphia, PA). The following was the synthetic cycle for the addition of each new residue. The t-BOC group on the growing peptide chain was removed with 45% trifluoroacetic acid (TFA) and 5% anisole (as a scavenger to minimize possible Trp oxidation) in dichloromethane (DCM). After neutralization with 10% diisopropylethylamine (DIPEA), the next t-BOC blocked amino-acid, added in approx. 6-fold excess, was coupled for approx. 2 h with 6.5% diisopropylcarbodiimide (DIPCI) in dimethylformamide (DMF). To minimize its acid destruction, the indole of tryptophan was protected by a formyl group. An amino-acid was coupled twice within one cycle in the case of bulky amino-acids, such as Val or Leu, or when the same amino-acid was linked consecutively. When Asn or Gln were added to the growing chain, 1.5 equivalents of 1-hydroxybenzotriazole hydrate (HOBt) was mixed with
the t-BOC amino-acid to minimize possible racemization. Unreacted growing chains with remaining free N-terminal amino groups were capped by reaction with 25% acetic anhydride in DCM. To add the next amino acid, the t-BOC group from the most recently added residue was removed by treatment with TFA and the synthesis cycle repeated. Upon completion of the chain, the final t-BOC group was removed and the N-terminal amino group acetylated with acetic anhydride. The resin bound peptide was then washed with methanol and stored at -20°C. To complete the synthesis, we removed the acid-labile side-chain protecting groups and cleaved the peptide from the resin with a trifluoromethanesulfonic acid/trifluoroacetic acid protocol [23]. Finally, we removed the formyl protecting group with a procedure supplied by Applied Biosystems [24]. Each peptide was purified first by passage over Sephadex G-10 equilibrated with 0.1 M ammonium acetate and then by HPLC with a semipreparative Ci8 reversed phase Dynamax Column (0.46 × 25 cm, 5 lz silica, 300 A pores, Rainin Instrument, CA) with acetonitrile/HzO gradients containing 0.1% trifluoroacetic acid. Peptide homogeneity was determined as a single peak in the HPLC. Peptide identity was verified by amino-acid composition and FAB-MS, both performed in The Ohio State University Campus Chemical Instrument Center. We also obtained UV spectra to verify that each peptide contained one Tyr and one Trp [25]. All CD measurements were taken with a JASCO J-500C spectropolarimeter (JASCO, Easton, MD) equipped with a water-jacketed 1-cm pathlength quartz cell maintained at +0.1°C over the range of 0-70°C. We determined the peptide concentration by measurement of the tyrosine plus tryptophan UV absorbance [25]. The CD instrument was calibrated with both ammonium-d-camphor-10-sulfonate [26] and o( - )pantolactone [27]. The pulse radiolysis procedures were those described elsewhere [15]. Rate constants were obtained by nonlinear fitting of absorbance-time profiles using the data acquisition package ASYST (Macmillan Software, New York, NY). All other nonlinear fits were done with either MINSQ (MicroMath Scientific Software, Salt Lake City, UT) or with a code developed in this laboratory from the GRIDLS subroutine of Bevington [28]. Using standard procedures, we redistilled all the solvents and reagents used in the peptide synthesis (DCM, DMF, DIPEA and DIPCI). The remaining chemicals were used as purchased: TFA (protein sequencing grade), anisole, acetic anhydride, HOBt and thioanisole from Aldrich (Milwaukee, WI); ethanolamine and trifluoromethanesulfonic acid from Sigma (St. Louis, MO). The CD standards were supplied by JASCO (ammonium-d-camphor-10-sulfonate) and Aldrich (o-(-)-pantolactone). All solutions were pre-
288 pared with water obtained from a Millipore Milli-Q apparatus.
.000
(a)
-.0Ol -.002
Results and Discussion L
LRET in leucine zipper peptide Our overall goal is to study the properties of the L R E T process in distinct structural elements of proteins. In this study, we sought definitive evidence that L R E T can occur in a helix. For this purpose we constructed a leucine zipper-type peptide. An apparently essential feature for the leucine zipper structure, a dimeric a-helical coiled coil, is an amino-acid sequence in which every seventh residue is a leucine (or related residue) with interspersed apolar residues forming a 4 - 3 apolar residue repeat [29,30]. When folded into an a-helix, these apolar 4 - 3 repeat residues align along the helix surface, thus, stabilizing a coiled-coil dimer with the 4 - 3 apolar residues of one chain in contact with those of the other. O'Shea and co-workers [17-19] have shown by both CD and I H - N M R measurements that a synthetic 33-residue peptide (GCN4-pl), taken from the yeast D N A binding transcriptional regulator GCN4, forms a stable parallel dimer of a-helices. Using G C N 4 - p l as a model we constructed a 33-met we call GCN4-6. Its structure is given below: AcetyI-AVKQLEDKVWELLNKNYKLENEVAKLKK-
-.003
"6 -.O04
X~
-,11115
-.006 270
I 280
I 290
I 300
I 310
i 320
I 330
I 340
Temp. (K) .000
(b) -.001 -.002
"~
-.11114
"O
-.005
-.006 270
280
I 300
290
I 310
I 320
I 330
I 340
Temp. (K)
Fig. l. Helix-coil transition with temperature for GCN4-6. Ellipticity was measured at 222 nm under the experimental conditions, 24/~M GCN4-6 (pH 7.0), 5 mM phosphate buffer, 1.0 M NaCI. (a), Data fit to equation generated from the three state equilibrium of Eqn. 7 in the text. Residuals are plotted in the inset; (b), data fit to equation generated from the two state-equilibrium of Eqn. 6 in the text.
LVAEA (The number 6 indicates that Tyr and Trp are separated by six intervening residues). In the design of GCN4-6 we replaced the following residues of GCN4pl: R1A, M2V, E10W, S14N, H18K, R25K, G31A, R33A. l Base on the proposed coiled-coil dimeric structure of G C N 4 - p l , the aromatic side-chains of the T y r O H / T r p " redox pair (in the 1-electron oxidized form of GCN4-6) should face into the aqueous environment with their fl-carbons separated by very nearly 10 A along a line parallel to the helix axis. We synthesized GCN4-6 using the methods described earlier. Its helical content, estimated from its ellipticity at 222 nm, is concentration-dependent at p H 7 (5 mM phosphate buffer), I°C and low salt ( < 0.2 M NaCI). At 1.0 M NaCI the helical content is independent of peptide concentration over the range 5 - 3 0 jzM and at I°C is roughly 70-80%. However, even at high salt, the apparent Tm for the helix-coil transition is dependent on the peptide concentration. These observations are consistent with a helical structure induced
i The changes introduced into the GCN4 peptide are indicated by a standard procedure, the one-letter abbreviation of the original amino acid followed by the residue position and the one-letter abbreviation of the new amino acid. For example, R1A indicates that Arg at position 1 has been replaced by an alanine.
by peptide aggregation. Assuming the simplest dimer model for the leucine zipper, we analyzed the melting curve of ellipticity vs. temperature on the basis of a two-state process in which the random coil m o n o m e r (Mrc) associates directly to the helical dimer ( D h ) . 2Mrc ~ Dh
(6)
The fit of the equation generated by this model to the observed data is adequate (Fig. lb), indicating that the random coil monomer in equilibrium with an ahelical dimer is a reasonable interpretation. However, there are small but unmistakable deviations between the data and the calculated best fit curve. We, therefore, considered the next more complex, possibility, a three-state model in which the m o n o m e r exists in both helical (M h) and random coil forms. 2Mrc ~ 2M h ~ D h
(7)
The three appropriate equations associated with this scheme, one mass balance and two equilibrium expressions, arc." PT = 2 ( D h ) + (Mh) + ( M r s ) Kh~I = ( M h ) / ( M r c ) = exp( - A H h d / R T + AShc I / R ) Kass = ( D h ) / ( M h ) 2 = exp( --
AH~/RT+
ASass / R )
(8)
289 PT is the total peptide concentration, ghe I the equilibrium constant for the m o n o m e r coil to helix transition and Kass the m o n o m e r helix to dimer association constant, The equilibrium constants have also been expressed in terms of their standard enthalpies and entropies. This set of nonlinear equations can be rearranged to quadratic expressions for the concentrations of the individual species. As an example, the concentration of the helical m o n o m e r is given by:
-.,
0~" '0
,
+ ~'~""~ + .."" - " ' ~... _'" ~ +" ~
10000
•
-
2P T
(9)
with similar quadratic expressions for D h and Mrc. The mean residue ellipticity is related to the concentrations of the three species by: O = [O]M~(Mrc) +2[O]oh(Dh) + [O]M.(Mh)
40
o...monomer 1000
0
.' "
"'"'--.
60© 3.00
'
20
0
•"
0
"~
~
:1.14
*.28
' 1.4t
~ 3.56
Q
0 $.70
1/T (K'lxl0))
ASas s / R )
exp(AHass / R T - ASass/R)= 0,
00
(2-.
.~
2(Mh) 2 + (Mh)[1 + exp(A Hh~I / R T - SheI/R)]
× exp(AHa,s/RT -
100
(]o)
where the [O]Mrc, [O]Mh, [O]Dh are the molar mean residue ellipticities of the random coil monomer, the helical m o n o m e r and the dimer, respectively. Assuming that [O]Dh=[O]Mh, we fit Eqn. 10 plus the three quadratic expressions (9) to the experimental data. In this nonlinear regression analysis based on the G R I D L algorithm of Bevington [28], the constants left free to vary were [O]M,, [O]M,c, AHhet, AShel, AHass and ASas s. As seen in Fig. la, this model yields a reasonable fit. We, therefore, suggest that while the simpler d i m e r / m o n o m e r model of Eqn. 6 describes the essential features of the h e l i x / r a n d o m coil transition in CGN4-6, an additional perturbation must be included in the final scheme. Whether this correction is sufficient for the complete determination of the aggregation process must await higher precision data. Thus, formally, we still have only an incomplete understanding of the GCN4-6 melting behavior. Nevertheless, the CD data clearly indicate that: (i), in solution near 0°C and at sufficiently high concentrations GCN4-6 occurs predominantly as an aggregate, most likely as a dimer, in which; (ii), each chain has a helical content of 7 0 - 8 0 % (The fit to the model of Eqn. 7 also suggests that those chains which are monomeric at this low temperature will be predominantly helical.) and (iii), at the higher temperatures investigated the chains exist as random coils. Finally, the diffraction pattern obtained from crystals of GCN4-6 is consistent with the coiled-coil parallel dimer structure of the leucine zipper reported for G C N 4 - p l (communication from M. Sundaralingam). We then looked for the L R E T process of Eqn. 4 in the GCN4-6 helical dimer, i.e., the reduction of T r p ' by a tyrosine side-chain when these redox partners are held apart by an a-helix. Our approach was to assume that the L R E T rate in a helical structure would not be
Fig. 2. Temperature dependence of electron transfer in GCN4-6, (o), GCN4-6: measured electron transfer rates under experimental conditions as in Fig. 1, except for the addition of 0.01 M N3 . (+), 17(Aib)-6, experimental conditions are those of Fig. 6. (. . . . . . ), monomer (%) calculated from constants obtained in the fit to the three-state model of Fig. 1. ( . . . . . . ), dimer (%) calculated from constants obtained in the fit to the three-state model of Fig. 1. identical to the electron transfer rate in a nonhelical structure. Thus, the t e m p e r a t u r e dependence of the L R E T process would reflect the electron transfer activation energies in both structures as well as the helixcoil transition enthalpy. Were this the case, then the Arrhenius plot (In kapo vs. 1/T) would be nonlinear over the t e m p e r a t u r e range of the helix-coil transition. To measure the electron transfer of reaction (4) in GCN4-6, we used the pulse radiolysis procedures described elsewhere [15]. The electron transfers we did observe were intra-, not intermolecular, since the bimolecular process makes a negligible contribution at the low peptide concentrations employed in these experiments [15]. As seen in Fig. 2, the Arrhenius plot (In kapp vs. 1/T) obtained with GCN4-6 is not linear. Moreover, there is an obvious transition in the electron transfer process, a transition that coincides with helixto-coil melting. Since GCN4-6 is helical at low temperatures (see above), we shall conclude that electron transfer in GCN4-6 at low temperature occurs across the helix structure and, therefore, must be a long range process. However, before reaching this conclusion we must consider another possible scheme. Were electron transfer only possible in the random coil and not the a-helical structure, then the observed electron transfer at low temperatures would be due to that small amount of random coil in equilibrium with the a-helical structure. Trp '-(helix)-TyrOH------//---,TrpH-(helix)-TyrO" ][fast
Trp -(coil)-TyrOH
fast
l[fast
(11)
, TrpH-(coil)-TyrO
This possibility derives from the observations that the peptide is not completely helical at low temperatures, as measured by CD and that linear extrapolation from
290 the high-temperature data to the low:temperature region would suggest an electron transfer in the random coil almost one order of magnitude faster than the electron transfer observed in the putative a-helical dimer. However, the reaction scheme of Eqn. 11 is not consistent with the data. Were the nonreactive helical structure in slow (relative to the rate of electron transfer) equilibrium with the reactive random coil, or were the conversion of helix-to-coil rate determining, then in the temperature region of the transition, where both random coil monomer and helical dimer must be present, we should observe a biexponential process. The faster phase would be from those random monomeric chains in the equilibrium established before the start of the reaction; the slower phase would arise from the slower obligatory conversion of the non-reactive a-helix to the reactive random coil. Additionally, the relative magnitudes of the two phases should be temperature dependent, the slower phase accounting for most of the reaction at low-temperatures and the faster accounting for most of the reaction at the high. At no temperature did we observe two phases of Trp" reduction. Fig. 3 presents, as one example, the results of a kinetic experiment at 24°C, a temperature at which there is approx. 50% helical structure. This example shows the initial oxidation of Trp by N 3 as seen by the fast rise in absorbance at 510 nm due to the formation of T r p - The subsequent slower decay of Trp" back to Trp is due to electron transfer from the electron donor, the tyrosine side-chain. (The absorbance at 510 nm does not decay back to zero because of the finite equilibrium between the tyrosine and tryptophan radicals [15].) The experimental points have been fit by least squares to a biexponential equation; the first phase representing Trp oxidation, the second Trp" reduction. The good fit indicates that there is only one exponential process associated with the reduction of Trp" by tyrosine.
5.50
4,30
0
3.10
o
1.90
0.70
-0.50 0.00
020
0.40
0.60
0,80
1 O0
Time (msec) Fig. 3. Electron transfer in GCN4-6. Absorbance measured at 510 nm as a function of time under experimental conditions identical to those for the GCN4-6 data of Fig. 2. (o), Experimental points; ( ), best-fit calculated time-dependence based on the equation A = a 0 + a 1 e x p ( - k i t ) + a 2 e x p ( - k2t)
Therefore, for the scheme of Eqn. 11 to apply chain unfolding would have to be fast relative to electron transfer in the random coil. In this case, were the chain unfolding enthalpy comparable to or less than the activation energy of electron transfer in the random structured chain, then we would not expect the large nonlinearity observed (see below). On the other hand, were the unfolding enthalpy higher than the activation energy of electron transfer in the random chain, then the low-temperature region of the Arrhenius plot should be either linear (not concave up) or in a more complex (e.g., different steps in the unfolding process being rate determining at different temperatures) situation concave down. That the low-temperature data points turn upwards is evidence for a low-temperature form in which electron transfer is slower than that expected in the random coil existing at the same temperature, with both structures in rapid equilibrium. Since the predominating structure at low-temperature is the a-helical structure, this low rate constant must reflect electron transfer in the dimer. There is one last consideration. The CD data suggest that the chains within the dimer are not completely helical. The most likely interpretation is chain unravelling at the two ends. However, the redox d o n o r / a c c e p t o r pair is located well within the center of the helical structure, so that one cannot postulate direct contact between donor and acceptor due to chain flexibility at the two ends. Therefore, since there is electron transfer in GCN4-6 dimer at low temperature and, since T r p and Tyr are separated by a helical structure that does not permit contact between them, electron transfer must occur across the helix structure and must be a long range process. The estimated linear extrapolation from higher to lower temperatures suggests that the L R E T rate in the leucine zipper is almost one order of magnitude slower than the electron transfer rate in the random coil at the same low temperature. How is this explained? It is possible that the helical structure of GCN4-6 slows electron transfer because of some structural or geometric feature yet to be explained. However, electron transfer in the random coil could occur by direct contact between the donor and acceptor residues, a potentially faster process than LRET. Were this the predominant path of electron transfer in the random coil, then the mechanism of electron transfer would be different in the two structures, contact in the random structure and L R E T in the helical. At present, therefore, we cannot directly compare electron transfer rates in the two structures of GCN4-6. We can, however, note interesting comparisons in related peptides. Thus, at I°C, a temperature at which GCN4-6 is helical, the L R E T rate between Tyr and Trp" separated by an extended - p r o 3- chain is approx. 10-fold slower [31] than the rate across the a-helical GCN4-6 spacer. The
291 o
through space distance in both cases is approx. 10 A, but the through backbone distance in GCN4-6 is roughly twice that in Trp-(pro)3-Tyr. On the other hand, at 25°C the observed rate constants for LRET between TyrOH and Trp" separated by oligoglycine (12,15), oligoglutamate (15) and the 17(Aib)-n peptides (see below) with only a few exceptions fall into the range of (1-5). 104 s -1 and show only a negligible dependence on chain length. This is in contrast to the results we have obtained with the proline spacer (37).
(a)
lS 0
E ~
10-
% 0 ID "~
5-
0
(b)
% X
i
i
i
~
~
i
15"
Electron transfer in the peptide 17(Aib)-n We have also looked for electron transfer in a series of peptides based on the 17-residue design of Marqusee and Baldwin [16]. The a-helices of these peptides may be stabilized by glutamate and lysine i + 4 ion-pair interactions and by a favorable interaction between the charges on the chain and the helix dipole. In the peptides we synthesized, we varied the number of residues between the Trp and Tyr, with Tyr located at the C-terminal end of the chain. A typical peptide with 7 intervening residues is: AcetyI-AEUAAKEUAWAKEUAAKY-NH 2 U is the one-letter abbreviation for a-methyl alanine (2-amino isobutyric acid) also abbreviated as Aib. For convenience we have named this illustrated peptide 17(Aib)-7; the first number refers to the number of residues in the peptide and the second to the number of residues between the Trp and Tyr. Because of steric interactions, the ~b,y angles associated with the Aib residue are constrained to a region of the Ramachandran map that corresponds to the a-helix or the 310helix and small Aib containing peptides contain either of these structures (see Refs. 32,33 and references therein). We, therefore, included Aib residues in an attempt to stabilize further the helical content of the peptides synthesized for this study. Our peptides had 3, 6, 7, 8, 11 and 13 intervening residues. In each case Trp was substituted for either Ala (n = 6, 7, 11) or Aib (n = 3, 8, 13). All the synthetic 17(Alb)-n peptides were at least partially a-helical at I°C, as seen from the characteristic CD spectrum with troughs located at 208 and 222 nm. We estimated the helicity of each from the 222 trough by assuming 3000 deg cm z dmo1-1 for the molar residue ellipticity of the random structure and calculating an a-helical content with the procedure of Chen et al. [34]. Each peptide, except 17(Aib)-3 with a greater apparent helicity, has an apparent helical content near 60% (range, 57-64%; average, 60.2 + 2.3%) at 1°C, F < 0.1, pH 7.0. (Since the ellipticity does not reach a constant value as the temperature approaches 0°C, we cannot determine whether 60% of all the peptide chains are completely helical, with the remain-
i
S-
~
1'0
1'~
pH Fig. 4. Helical content of monomeric peotides as a function of pH. Ellipticity at 222 nm measured at I°C in a solution containing 1 mM sodium citrate, 1 mM sodium borate, 1 mM sodium phosphate dibasic and 0.01 M NaCl; pH adjusted with NaOH or HCI. Top, 17(Ala)-6; bottom, 17(Aib)-7.
ing 40% in a coil form, or whether all of the chains contain both structures and, thus, are 60% helical and 40% coil.) This helical content is independent of concentration over a range of 8-30 I~M. While the helicities of the peptides synthesized in Baldwin's laboratory are higher, those of the 17(Aib)-n peptides may be consistent with the reported helix destabilization by Trp [35]. The measured ellipticities, of all the 17(Aib)-n peptides: (i), depend on pH (Fig. 4) - constant between pH 2 and 8 and decrease at higher pH); (ii), decrease with increasing NaCI concentration; and (iii), decrease with increasing temperature. In analyzing helix temperature dependencies we assumed a noncooperative two-state model, with a simple equilibrium between helical and nonhelical structures. Consistent with this model, we obtained linear van't Hoff plots in each case. The estimated unfolding enthalpies were approx. 16 kJ/mol (range 14-20 kJ/mol; avg. 16 +_ 0.3 kJ/mol). These enthalpies are consistent with those reported by Merutka and Stellwagen [35], who worked with closely related peptides, but are a good deal lower than the approx. 42 kJ/mol reported later from this same laboratory [36]. We should note that while consistent with the simple two state melting model used in the fitting, the collected data cannot rule out a cooperative transition. Thus, the computed values of the unfolding enthalpies would be sensitive to the validity of the model on which the fitting was based. Our reported enthalpies are only preliminary estimates. While we included Aib in order to stabilize the a-helical structure of the synthesized peptides, there appears to be little stabilization at neutral pH. 17(Aib)6 and 17(Ala)-6, in which the three Aib residues were
292 replaced with Ala, had the same a-helical contents, 61% and 62%, respectively. Moreover, the unfolding enthalpy, 20 kJ/mol, of 17(Ala)-6, is within experimental error, that of the average for all the Aib peptides. However, while the Aib peptides maintain a constant helicity as the pH is lowered below 7, the helicity of 17(Ala)-6 decreases at lower pH (Fig. 4). Thus, at acidic pH, Aib does appear to stabilize a helical structure. While we cannot yet explain this apparent pH dependence of Aib helix stabilization, these results suggest that the Aib contribution to the stabilization of helices is not a simple linear (i.e., additive) property. With each of the synthesized 17(Aib)-n we measured electron transfer rates as a function of temperature. As in the GCN4-6 case, we expected the Arrhenius plots for the measured first-order intramolecular rate constants to be nonlinear. Once again the low concentrations of the peptides insured against a significant bimolecular reaction. At approx. 1°C, pH 7, in 5 mM phosphate buffer plus 0.01 M NaN 3 (added for the initiation of the electron transfer) the apparent first order electron transfer rate constant in the 17(Aib)-n peptide series increases from 1.9- 103 s- i in 17(Aib)-13, to 17 • 103 s - t in 17(Aib)-3. The observed rate constant dependence on amino-acid separation (Fig. 5) appears to be exponential, as would be predicted by Marcus theory for an L R E T process [37]. For comparison, Fig. 5 also contains earlier distance dependence data for T y r O H / T r p electron-transfer across the oligoproline spacer [38]. The inflexibility of the proline residue insures that the T y r O H and Trp" residues cannot come into contact and electron transfer must be long range. With the oligoproline spacer L R E T is relatively insensitive to the separation distance; i.e.,/3ap o, the slope of the In kap o v s . distance curve, may be as low as 0.27 ,~-l. Were electron transfer in 17(Aib)-n an L R E T process, then the apparent distance, in terms of the peptide main chain, dependence of the rate constant would have to be even shallower.
{:3 10000
'0 10OO
100
I
L
2
&
I
i
4
number
i
i
6
; 8
of
i
I
10
i
~
12
i
14
residues
Fig. 5. Electron transfer rate constant dependence on 17(Aib)-n chain length. (13 13), 17(Aib)-n peptides at approx. I°C, pH 7, 5 m M phosphate buffer, 0.01 M NAN3; ( zx zx), Tyr-(Pro)n-Tr p at 25°C. Data taken from Ref. 38.
100000
O
i
0
10000
O
3000 2.80
I
i
i
I
~.oo
3.=o
3.40
3.80
lIT
(K"xI0
3.80
~)
Fig. 6. Temperature-dependence of electron transfer rate constant with 17(Aib)-7. Approx. 0-60°C, pH 7, 5 mM phosphate buffer, 0.01 M NaN 3.
We also determined the temperature dependence of the electron transfer rate constant with each of the 17(Aib)n peptides. Over the range of approx. 0-60°C the helix content of each peptide decreases from approx. 60% to approx. 10% as measured by CD. Unlike the GCN4-6 experiment, the Arrhenius plots were all linear over this temperature range where the peptide helices unravel. Example data for 17(Aib)-7 are in Fig. 6 and for 17(Aib)-6 in Fig. 2. The estimated activation energies obtained from all these linear Arrhenius plots are near 23 k J / m o l (range 21-25 kJ/mol; avg. 23 + 1.7 kJ/mol). This is similar to the L R E T E a of approx. 21 k J / m o l found when this same amino-acid pair is separated by the oligoproline spacer [38]. Moreover, the electron activation energies found with Trp-Tyr, 21 k J / m o l [12]; Tyr-Trp, 22 k J / m o l [38] and Tyr-Glu-GluTrp, 21 k J / m o l [16], are also closely similar. While one could argue that there is L R E T across an a-helical structure at the lower temperatures even though the Arrhenius plots obtained with the 17(Aib)-n peptides are linear, there is another interpretation for the observed linearity. Were electron transfer sufficiently slow in the a-helix and were the equilibrium between helix and coil sufficiently fast, then electron transfer need not occur in the helical structure at all. This explanation would be represented by a scheme similar to that of Eqn. 11. There is initial fast unfolding followed by electron transfer in the nonhelical form, but there could also be a slower L R E T in the helical form. In the GCN4-6 case, we ruled out an initial unfolding scheme by appealing to kinetic arguments and to the shape of the nonlinear Arrhenius plot. On the other hand, the unfolding enthalpies obtained from the CD dependencies of the 17(Aib)-n peptides are less than 16 + 0.3 k J / m o l , though close to the E a we measured for the electron transfers. Hence, we would be reluctant to conclude that we have observed an L R E T process in the 17(Aib)-n peptides. Comparing the GCN4-6 and 17(Aib)-6 data (Fig. 2) allows further interpretation of the 17(Aib)-n data.
293 Above 25°C, the rate constants for electron transfer in GCN4-6 coincide with the rate constants for electron transfer in 17(Aib)-6. The two peptides are analogous in that 6 residues separate Trp" and T y r O H in both. Because the CD results suggest that both 17(Aib)-6 and GCN4-6 are largely random monomers at these higher temperatures, we argue that the coincidence of the electron transfer rates is not coincidental. Rather, the rate constants in both peptides at these higher temperatures must be generic and associated with electron transfer in the nonhelical structure. Below 25°C, the rate constants obtained with GCN4-6 are clearly lower than those obtained with 17(Aib)-6. The linearity of the 17(Aib)-6 results together with the difference of rates between 17(Aib)-6 and GCN4-6 at low-temperatures supports the proposed initial unfolding reaction scheme. We conclude that electron transfer in the 17(Aib)-n series of peptides at all measured temperatures occurs primarily through the nonhelical structure. However, whether electron transfer in the random structure occurs by direct contact between electron donor and acceptor or is long-range hinges on whether the rate of d o n o r / a c c e p t o r 'collision' is rapid or slow relative to the observed electron transfer. With respect to this question, there are at least two observations to be considered. First, previous calculations [39,40] have shown that the &,~ dihedral angles permitted for the Aib residue occur within two restricted regions on the Ramachandran plot - near - 5 7 °, - 4 7 ° and +57 °, +47 °. In these two regions are found right and left handed a-(or 310-)helices respectively. This explains why the Aib residue should help to stabilize a helical structure. However, this steric restriction on rotation about the N-C ~ and the C'~-C backbone bonds must make peptides that contain Aib less flexible than those that do not. There is one Aib residue between Tyr and Trp in 17(Aib)-6; there are no Aib residues in GCN4-6. Yet the rates of electron transfer are closely similar in the two 'random' coils. Second, electron transfer in the 17(Aib)-n series is exponentially dependent on the number of amino-acid residues in the chain between donor and acceptor. Is this dependence, which is consistent with an L R E T mechanism, also consistent with a contact electron transfer mechanism? In conclusion, we have argued for an L R E T process in the a-helical structure of the GCN4-6 dimer. Moreover, while the rate of electron transfer appears to be slower in the helix than in the random coil, we cannot directly compare the two, for we do not know whether electron transfer in the random structure is a contact or long range process. Other laboratories have also reported electron transfer across helical structures [41-43]. However, there was no careful consideration in these reports of whether the local structures over which electron transfer occurred were indeed helical. Our results suggest that conclusions of L R E T across
helices must be considered carefully, since electron transfer could occur after rapid melting to an unstructured form of the peptide.
Acknowledgements We are indebted to Edward Ray who has maintained our linear accelerator permitting the pulse radiolysis experiments described. This work was supported by grant GM 35718 from the National institutes of Health.
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