- Email: [email protected]
Design, synthesis and characterization of a water-soluble β-sheet peptide
Design, Synthesis and Characterization of a Water-soluble p-sheet Peptide David S. Wishart, Les H. Kondejewski, Paul D. Semchuk, Cyril M. Kay, Robert S, Hodges, and Brian D. Sykes Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
I. Introduction Peptide models of a-helices have revealed much about the intrinsic and extrinsic factors that control helix formation in peptides and proteins (1-5). While considerable progress has been made in our understanding of helix formation and stabilization, the same cannot be said of the situation regarding two other important classes of secondary structure: p-sheets and p-tums. The reason for this is that there has not yet been a p-sheet model developed that is as simple to prepare and as easy to characterize as a monomeric helix. Early peptide models of p-sheets were typically based on large homooligopeptide aggregates (6), which were too poorly defined to be of any practical use in determining p-sheet propensities. More recent work involving diacylaminoepindolidione or dibenzofuran-propionate mimetics of p-sheets and p-turns (7, 8), while very promising, are also problematic because of their reliance on non-peptide constituents, their difficulty in preparation, and in the case of the epindolidione model, their limited solubility. As an alternative to the peptidomimetic approach to studying p-sheet propensities, two protein-based models have recently been proposed. One is based on the Bl domain of staphylococcal protein G which binds IgG and is termed GBl (9, 10); the other is based on the zinc-fmger peptide CP-1 (11). While the results from the zincfinger peptide work agree quite well with published statistical p-sheet propensities, they unfortunately do not agree with the results from the GBl work (9). The poor agreement between the two models may result from neither model being a pure p-sheet. Consequendy, in both systems contributions from helices, metal ions and other p-strands may affect the measured p-sheet propensities in ways that are difficult to characterize. Given the limitations of the above systems, it is apparent that the optimal peptide model of a p-sheet (and a p-turn) should be as analagous to the monomeric helix models as possible. In particular, the ideal p-sheet model should be small (< 20 residues), monomeric, water-soluble, pure (composed of only p-sheets and p-turns), amphipathic (to investigate sidedness), reversibly denaturable, composed of only natural amino acids, easily synthesized and easily characterized by standard spectroscopic techniques. We believe that we have developed such a peptide model. It is based on the naturally occurring cyclic peptide gramicidin S, an antibiotic produced by the bacterium bacillus brevis (12). The schematic structure of gramicidin S as determined by X-ray and NMR studies (13, 14) is shown in Figure 1. TECHNIQUES IN PROTEIN CHEMISTRY VI Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
451
452
David S. Wishart et al.
D-Phe
D-Phe
Om
Figure 1. The structure of gramicidin S from bacillus brevis. Note the well-defined p-pleated sheet structure.
Gramicidin S is a symmetric decapeptide (sequence: Val-Om-Leu-dPhe-Pro-ValOm-Leu-dPhe-Pro) composed of two antiparallel p-strands linked by two type IF p-turns. In this report we will describe methods that we have developed to easily prepare a number of gramicidin S (p-sheet) analogs using solid phase peptide synthesis and a simple but effective cyclization protocol. We will also present data describing the effects of selected residue substitutions on the solubility and structural stability of these peptides. Furthermore, we will demonstrate how lengthened (12 residue) analogs of gramicidin S exhibit features of cold denaturation and trifluoroethanol (TFE)-induced structure formation. These results will be used to demonstrate the potential of this system for determining p-sheet and p-turn propensities of amino acids and for studying the influences of hydrophobicity, sidechain packing, charge, temperature and solvent on p-sheet formation.
11. Materials and Methods A. Synthesis
and Cyclization
of Gramicidin
S and Analogs
Most syntheses of gramicidin S reported to date (12) are based on tedious solution phase approaches that take up to two weeks to complete. Because of the number of analogs that we anticipated using in this study, it became essential to develop a facile, semi-automated approach. The protocol described below allows gramicidin S analogs of varying length and composition to be synthesized in high yield and purified to homogeneity in less than three days with minimal human intervention. Beginning with either Boc--Lys (CL-Z)-PAM or Boc-Orn (CL-Z)-PAM resin, the following 10-residue linear peptides were synthesized using an Applied Biosystems 430A automated peptide synthesizer. Gramicidin S: LFPVOLFPVO LYPVKLYPVK Peptide 1: LSPVKLSPVK Peptide 2: Peptide 3: LNPVKLNP VK Peptide 4: LHPVKLHPVK
(D-Phe ~> D-Tyr) (D-Phe ~ > D-Ser)
(D-Phe - > D-Asn) (D-Phe - > D-His)
A Water-Soluble P-Sheet Peptide
453
The following 12-residue peptides were synthesized in the same manner, starting with Boc-Val-PAM resin, but with the £-amino group of lysine protected with Fmoc (instead of Cl-Z): Peptide 5:
KLKFPKVKLFPV
Peptide 6: Peptide 7:
ILKSPKVILSPV GLKSPKVILSPV
It should be noted that substitution of lysine to ornithine has no effect on the structure or activity of the peptide (12). The blocked peptides were cleaved from the resin using anhydrous HF in the presence of anisole. All peptides were purified prior to cyclization using reversed phase (Cg) HPLC with a linear AB gradient, where A=0.05% TFA/H2O and 3=0.05% TFA/acetonitrile. Cyclizations were performed at concentrations of --2 mg/mL in dichloromethane using 1.2 equivalents each of N,N'-dicyclohexylcarbodiimide, N-hydroxybenzotriazole and diisopropylethylamine. The progress of the cyclization reaction was monitored by both reversed phase HPLC and plasma desorption TOP mass spectrometry. For gramicidin S and Peptides 1-3, cyclizations were typically complete in 6 hours, with final overall yields ranging from 45-90%. These high yields were achieved with no indication of racemization, while using only the free peptide as the starting material. Peptides 4-7 took longer to cyclize (~24 hrs.) and the overall yields tended to be lower (5-10%). Fmoc groups on Peptides 5, 6 and 7 were removed after the cyclization step by treatment with piperidine (2 hr). Final purification was achieved using reversed phase HPLC. B. Spectroscopic
Characterization
(NMR and CD)
All NMR spectra were collected on a Varian Unity 500 MHz spectrometer (^H frequency 499.8 MHz) equipped with a 5 mm inverse detection probe. Sample concentrations were typically 1-2 mM and sample temperatures maintained at 25 "C (unless otherwise noted). Sample pH was typically 3.5 - 4.0. Onedimensional ^H data were acquired with a 'H sweep width of 6000 Hz and an acquisition time of 2.3 seconds. The residual water signal was suppressed by presaturation. 'H DQF-COSY, NOESY and TOCSY (15) spectra were collected and processed using standard methods. All chemical shifts were referenced relative to internal DSS. CD samples (-1 mg/mL) were prepared by dissolving the peptides into a 10 mM sodium acetate buffer (pH 5.5) and sonicating for approximately 1 minute. Insoluble material was removed by centrifugation. CD spectra were recorded at 25 *C (unless otherwise noted) on a Jasco J-500C spectropolarimeter using a 0.02 cm pathlength cell attached to a circulating water bath. CD spectra represent the average of four scans collected over a wavelength interval of 190 to 250 nm. Ellipticity is reported as mean residue ellipticity [9], with an approximate error of-500° at 220 nm.
III. Results A. Substitution
Effects
of D-Phe on Solubility
and
Structure
Gramicidin S is not readily soluble in water and often precipitates in the presence of divalent counter ions (HPO4). In order to design p-sheet analogs that were more water soluble and less sensitive to salt or pH, we investigated the effect of replacing the most hydrophobic amino acid (D-Phe) in gramicidin S with a series
454
David S. Wishart et al.
of polar amino acids. Analogs were synthesized with D-Tyr (Peptide 1), D-Ser (Peptide 2), D-Asn (Peptide 3) and D-His (Peptide 4) in the 4 and 4' positions (numbering according to reference 12). Three of the four peptides were found to be significandy more soluble than native gramicidin S, with Peptides 1 and 2 being soluble to >10 mg/mL and Peptide 4 being soluble to 8.5 mg/mL. In addition, all four peptides were analyzed by NMR and far UV CD spectroscopy to characterize their structure. For peptides 1, 2 and 3, chemical shifts, coupling constants, nOe connectivities and far UV CD spectra are all consistent with a psheet structure similar to gramicidin S. Peptide 4, however, appears to retain very littie p-sheet structure. These results are summarized in Figure 2, where the chemical shift index (16), derived from a-^H NMR chemical shifts, is plotted for each analog.
talLlllJ
tanuHiJ Peptide 3
Peptide 1
V K L N P V K L N P
V K L Y P V K L Y P
0)
ULiJL
o Peptide 4 V K L H P V K L H P
Figure 2. Chemical Shift Index plots of peptides 1, 2, 3 and 4. Arrows indicate the location of P-sheets in these peptides. Clusters of three or more positive chemical shift index (CSI) values are indicative of a p-sheet. Overall, these results suggest that it is possible to greatiy increase the solubility of this p-sheet model without significantly disrupting the structure. They also suggest that some residues (His in particular) can disrupt the type IF p-tum and eliminate most of the p-sheet structure. This result also implies that it may be possible to use host-guest techniques (17) to study type IF p-tum propensities with this system. B. Effects of Extending
the
p-sheet
An unexpected result concerning the 10 residue p-sheet analogs was their remarkable stability. None of the peptide models exhibited any significant structural change upon heating to 85 °C or upon addition of significant quantities of chaotropic solvents. To make these peptides more susceptible to denaturation, the p-sheet was extended by two residues. This chain extension was expected to increase the chain entropy, thereby reducing the thermal stability of the peptide. Unexpectedly, the addition of two lysines to the hydrophilic side of gramicidin S (Peptide 5) significandy reduced its p-sheet content under benign conditions. However, the addition of the structureinducing solvent trifluroethanol (TFE) actually enhanced the p-sheet content of this molecule (Figure 3a). While TFE is commonly used to induce helical
455
A Water-Soluble P-Sheet Peptide
Structure in peptides, we believe this represents one of the few instances where TFE has been used to induce the formation of p-sheet structure (18). A
5-1
_-^ o E •a u
0^ i"
•\
o E •o E o
^
''^^^
2.
'o -15H
\
'o
* TFE
B
5
-5
-10 -15
A ',\
5 "C
»\ * ^^
-25 J 190
200
220 230 Wavelength (nm)
240
250|
»C
-----
2! -20
_ 210
. ' • ' • *
' ^^ ^85
^^
X
5;-20
,"' ^ y' /
-25
1
190
200
210 220 230 Wavelength (nm)
240
250|
Figure 3. A) CD spectrum of Peptide 5 with TFE (51% P-sheet) and without TFE (26% Psheet). B) CD spectrum of Peptide 5 at 5 'C and 85 *C. Spectra were analyzed using the program RBOCON (R.F. Boyko, unpubUshed). Another interesting feature of this extended p-sheet model can be seen in Figure 3b, where we show the effect of temperature on the CD spectrum of Peptide 5. Curiously, when the sample temperature is decreased (to 5 °C), the spectrum takes on more of a "random coil" character (only 20% p-sheet); but, when the temperature is increased to 85 X , the spectrum exhibits significantly more psheet character (38% p-sheet). In other words, high temperatures induce structure and cold temperatures reduce structure. We believe that this represents an excellent example of cold denaturation (19), and it suggests that the thermodynamics of p-sheet formation may be more complex than currently appreciated. C. Stabilizing
and Destabilizing
Amino
Acid
Substitutions
To enhance the p-sheet content of Peptide 5, two of its lysines were exchanged for isoleucines. Isoleucine is known to have a stronger p-sheet propensity than lysine (20). However, because these changes were expected to reduce the solubility of the peptide, the two phenylalanines were exchanged for serines. The resulting construct was called Peptide 6. A second construct (Peptide 7) was synthesized wherein one of the isoleucine residues was substituted with a glycine. This substitution was predicted to reduce the p-sheet content of the peptide. In Figure 4 we compare the CD spectra of Peptides 6 and 7. As expected, the spectrum for Peptide 6 has substantially more p-sheet than Peptide 7 (31% p-sheet vs. 2% p-sheet). Indeed, the CD spectrum for Peptide 7 closely resembles that of a classic random coil (21). Furthermore, as judged by the overall shape of the CD curve, the spectrum for Peptide 6 appears to have slightly more p-sheet (31%) than Peptide 5 (26%), as expected. It is also worth noting that Peptide 6, just like Peptide 5, exhibits features of cold denaturation and TFE induced structure stabilization (data not shown). These results suggest that a model based on the sequence of Peptide 6 has many of the features required of an ideal p-sheet model.
Davids. Wishart^r a/.
456 5 1
w^
1
0Peptide 7. • " '
CM
^^"^
8" . 5 . CO 0
^-10-
- 1
1
5 ^
"
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^/^
Peptide 6
••
200
210
220
230
240
250
1
Wavelength (nm)
Figure 4. CD spectra of Peptide 6 and Peptide 7 collected at 25 'C under benign (aqueous) conditions.
IV. Conclusions This report describes our efforts at designing, synthesizing and characterizing a water-soluble p-sheet analog. We think that we have succeeded in designing a 12 residue cyclic peptide (Peptide 6) which satisfies most of the criteria required of a model p-sheet: it is small (< 20 residues), monomeric, water-soluble, pure (composed of only p-sheets and p-turns), mostly amphipathic, reversibly denaturable, composed of only natural amino acids, relatively easily synthesized and easily characterized by either CD or NMR. We plan to refine this model to enhance its amphipathicity and to improve its cyclization efficiency. Once this refinement stage is complete, we will begin to systematically investigate the influence of amino acid substitutions on both the hydrophilic and hydrophobic sides of this peptide. The resultant data will be used to extract specific p-sheet propensities for all 20 naturally occurring amino acids. In addition to this work on monomeric p-sheets, we are beginning to study dimeric p-sheets (psandwiches) by preparing a variety of disulfide-linked p-sheet analogs. This will allow us to investigate the influences of side chain packing and hydrophobic effects on the stabilization of "idealized" p-sandwiches. We are hopeful that these model systems will provide researchers with the detailed information they need to understand the intricacies of p-sheet and p-turn formation in natural proteins.
References 1. 2. 3. 4. 5. 6. 7.
Padmanabhan, S., Marquesee, S,, Ridgeway, T., Laue, T.M., and Baldwin, R.L. (1990) Nature 344, 268-270. O'Neil, K.T., and DeGrado, W.F. (1990) Science 250, 646-651. Zhou, N.E., Kay, CM., and Hodges, R.S. (1992) J. Biol. Chem. 267, 2664-2670. Hill, C.P., Anderson. D.H., Wesson, L., DeGrado, W.F., and Eisenberg, D. (1990) Science 249, 543-546. Horovitz, A:, Matthews, J.M„ and Fersht, A.R. (1992) J. Mol. Biol. Ill, 560-568. Hartman, R., Schawaner, R.C., and Hermans, J. (1974) J. Mol. Biol. 175, 195-212. Kemp, D.S, (1990) Trends Bioiechnol 8, 249-255.
A Water-Soluble P-Sheet Peptide 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
457
Diaz, H., Tsang, K.Y., Choo, D., and Kelly, J.W. (1993) Tetrahedron 49, 3533-3545. Minor, D.L., and Kim, P.S. (1994) Nature 367, 660-663. Smith, K., Withka, J.M., and Regan, L. (1994) Biochemistry 33, 5510-5517. Kim, C.A., and Berg, J.M. (1993) Nature 362, 267-270. Izumiya, N., Kato, T., Aoyagi, H., Waki, M., and Kondo, M. (1979) "Synthetic Aspects of Biologically Active Cyclic Peptides - Gramicidin S and Tyrocidines", Kodansha Ltd., Tokyo. Hull, S.E., Karlson, R., Main, P., Woolfson, M.M„ and Dodson, E.J. (1978) Nature 275, 206-207. Krauss, E.M., and Chan, S.I. (1982) J. Am. Chem. Soc. 104, 6953-6961. Wuthrich, K. (1986) "NMR of Proteins and Nucleic Acids", J. Wiley & Sons, New York. Wishart. D.S., Sykes, B.D., and Richards, P.M. (1992) Biochemistry 31, 1647-1651. Scheraga. H.A. (1978) J. Pure Appl. Chem. 50, 315-324. Sonnichsen, F.D., Van Eyk, J.E., Hodges, R.S., and Sykes, B.D. (1992) Biochemistry 31. 8790-8798. Privalov, P.L., Griko, Y.V., Venyaminov, S.Y., and Kutyshenko, V.P. (1986) J. MoL Biol. 190, 487-498. Chou, P.Y., and Fasman, G.D. (1974) Biochemistry 13, 211 -222. Johnson, W.C. (1990) Proteins: Struct. Funct. Genet. 7, 205-214.
I. Introduction Peptide models of a-helices have revealed much about the intrinsic and extrinsic factors that control helix formation in peptides and proteins (1-5). While considerable progress has been made in our understanding of helix formation and stabilization, the same cannot be said of the situation regarding two other important classes of secondary structure: p-sheets and p-tums. The reason for this is that there has not yet been a p-sheet model developed that is as simple to prepare and as easy to characterize as a monomeric helix. Early peptide models of p-sheets were typically based on large homooligopeptide aggregates (6), which were too poorly defined to be of any practical use in determining p-sheet propensities. More recent work involving diacylaminoepindolidione or dibenzofuran-propionate mimetics of p-sheets and p-turns (7, 8), while very promising, are also problematic because of their reliance on non-peptide constituents, their difficulty in preparation, and in the case of the epindolidione model, their limited solubility. As an alternative to the peptidomimetic approach to studying p-sheet propensities, two protein-based models have recently been proposed. One is based on the Bl domain of staphylococcal protein G which binds IgG and is termed GBl (9, 10); the other is based on the zinc-fmger peptide CP-1 (11). While the results from the zincfinger peptide work agree quite well with published statistical p-sheet propensities, they unfortunately do not agree with the results from the GBl work (9). The poor agreement between the two models may result from neither model being a pure p-sheet. Consequendy, in both systems contributions from helices, metal ions and other p-strands may affect the measured p-sheet propensities in ways that are difficult to characterize. Given the limitations of the above systems, it is apparent that the optimal peptide model of a p-sheet (and a p-turn) should be as analagous to the monomeric helix models as possible. In particular, the ideal p-sheet model should be small (< 20 residues), monomeric, water-soluble, pure (composed of only p-sheets and p-turns), amphipathic (to investigate sidedness), reversibly denaturable, composed of only natural amino acids, easily synthesized and easily characterized by standard spectroscopic techniques. We believe that we have developed such a peptide model. It is based on the naturally occurring cyclic peptide gramicidin S, an antibiotic produced by the bacterium bacillus brevis (12). The schematic structure of gramicidin S as determined by X-ray and NMR studies (13, 14) is shown in Figure 1. TECHNIQUES IN PROTEIN CHEMISTRY VI Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
451
452
David S. Wishart et al.
D-Phe
D-Phe
Om
Figure 1. The structure of gramicidin S from bacillus brevis. Note the well-defined p-pleated sheet structure.
Gramicidin S is a symmetric decapeptide (sequence: Val-Om-Leu-dPhe-Pro-ValOm-Leu-dPhe-Pro) composed of two antiparallel p-strands linked by two type IF p-turns. In this report we will describe methods that we have developed to easily prepare a number of gramicidin S (p-sheet) analogs using solid phase peptide synthesis and a simple but effective cyclization protocol. We will also present data describing the effects of selected residue substitutions on the solubility and structural stability of these peptides. Furthermore, we will demonstrate how lengthened (12 residue) analogs of gramicidin S exhibit features of cold denaturation and trifluoroethanol (TFE)-induced structure formation. These results will be used to demonstrate the potential of this system for determining p-sheet and p-turn propensities of amino acids and for studying the influences of hydrophobicity, sidechain packing, charge, temperature and solvent on p-sheet formation.
11. Materials and Methods A. Synthesis
and Cyclization
of Gramicidin
S and Analogs
Most syntheses of gramicidin S reported to date (12) are based on tedious solution phase approaches that take up to two weeks to complete. Because of the number of analogs that we anticipated using in this study, it became essential to develop a facile, semi-automated approach. The protocol described below allows gramicidin S analogs of varying length and composition to be synthesized in high yield and purified to homogeneity in less than three days with minimal human intervention. Beginning with either Boc--Lys (CL-Z)-PAM or Boc-Orn (CL-Z)-PAM resin, the following 10-residue linear peptides were synthesized using an Applied Biosystems 430A automated peptide synthesizer. Gramicidin S: LFPVOLFPVO LYPVKLYPVK Peptide 1: LSPVKLSPVK Peptide 2: Peptide 3: LNPVKLNP VK Peptide 4: LHPVKLHPVK
(D-Phe ~> D-Tyr) (D-Phe ~ > D-Ser)
(D-Phe - > D-Asn) (D-Phe - > D-His)
A Water-Soluble P-Sheet Peptide
453
The following 12-residue peptides were synthesized in the same manner, starting with Boc-Val-PAM resin, but with the £-amino group of lysine protected with Fmoc (instead of Cl-Z): Peptide 5:
KLKFPKVKLFPV
Peptide 6: Peptide 7:
ILKSPKVILSPV GLKSPKVILSPV
It should be noted that substitution of lysine to ornithine has no effect on the structure or activity of the peptide (12). The blocked peptides were cleaved from the resin using anhydrous HF in the presence of anisole. All peptides were purified prior to cyclization using reversed phase (Cg) HPLC with a linear AB gradient, where A=0.05% TFA/H2O and 3=0.05% TFA/acetonitrile. Cyclizations were performed at concentrations of --2 mg/mL in dichloromethane using 1.2 equivalents each of N,N'-dicyclohexylcarbodiimide, N-hydroxybenzotriazole and diisopropylethylamine. The progress of the cyclization reaction was monitored by both reversed phase HPLC and plasma desorption TOP mass spectrometry. For gramicidin S and Peptides 1-3, cyclizations were typically complete in 6 hours, with final overall yields ranging from 45-90%. These high yields were achieved with no indication of racemization, while using only the free peptide as the starting material. Peptides 4-7 took longer to cyclize (~24 hrs.) and the overall yields tended to be lower (5-10%). Fmoc groups on Peptides 5, 6 and 7 were removed after the cyclization step by treatment with piperidine (2 hr). Final purification was achieved using reversed phase HPLC. B. Spectroscopic
Characterization
(NMR and CD)
All NMR spectra were collected on a Varian Unity 500 MHz spectrometer (^H frequency 499.8 MHz) equipped with a 5 mm inverse detection probe. Sample concentrations were typically 1-2 mM and sample temperatures maintained at 25 "C (unless otherwise noted). Sample pH was typically 3.5 - 4.0. Onedimensional ^H data were acquired with a 'H sweep width of 6000 Hz and an acquisition time of 2.3 seconds. The residual water signal was suppressed by presaturation. 'H DQF-COSY, NOESY and TOCSY (15) spectra were collected and processed using standard methods. All chemical shifts were referenced relative to internal DSS. CD samples (-1 mg/mL) were prepared by dissolving the peptides into a 10 mM sodium acetate buffer (pH 5.5) and sonicating for approximately 1 minute. Insoluble material was removed by centrifugation. CD spectra were recorded at 25 *C (unless otherwise noted) on a Jasco J-500C spectropolarimeter using a 0.02 cm pathlength cell attached to a circulating water bath. CD spectra represent the average of four scans collected over a wavelength interval of 190 to 250 nm. Ellipticity is reported as mean residue ellipticity [9], with an approximate error of-500° at 220 nm.
III. Results A. Substitution
Effects
of D-Phe on Solubility
and
Structure
Gramicidin S is not readily soluble in water and often precipitates in the presence of divalent counter ions (HPO4). In order to design p-sheet analogs that were more water soluble and less sensitive to salt or pH, we investigated the effect of replacing the most hydrophobic amino acid (D-Phe) in gramicidin S with a series
454
David S. Wishart et al.
of polar amino acids. Analogs were synthesized with D-Tyr (Peptide 1), D-Ser (Peptide 2), D-Asn (Peptide 3) and D-His (Peptide 4) in the 4 and 4' positions (numbering according to reference 12). Three of the four peptides were found to be significandy more soluble than native gramicidin S, with Peptides 1 and 2 being soluble to >10 mg/mL and Peptide 4 being soluble to 8.5 mg/mL. In addition, all four peptides were analyzed by NMR and far UV CD spectroscopy to characterize their structure. For peptides 1, 2 and 3, chemical shifts, coupling constants, nOe connectivities and far UV CD spectra are all consistent with a psheet structure similar to gramicidin S. Peptide 4, however, appears to retain very littie p-sheet structure. These results are summarized in Figure 2, where the chemical shift index (16), derived from a-^H NMR chemical shifts, is plotted for each analog.
talLlllJ
tanuHiJ Peptide 3
Peptide 1
V K L N P V K L N P
V K L Y P V K L Y P
0)
ULiJL
o Peptide 4 V K L H P V K L H P
Figure 2. Chemical Shift Index plots of peptides 1, 2, 3 and 4. Arrows indicate the location of P-sheets in these peptides. Clusters of three or more positive chemical shift index (CSI) values are indicative of a p-sheet. Overall, these results suggest that it is possible to greatiy increase the solubility of this p-sheet model without significantly disrupting the structure. They also suggest that some residues (His in particular) can disrupt the type IF p-tum and eliminate most of the p-sheet structure. This result also implies that it may be possible to use host-guest techniques (17) to study type IF p-tum propensities with this system. B. Effects of Extending
the
p-sheet
An unexpected result concerning the 10 residue p-sheet analogs was their remarkable stability. None of the peptide models exhibited any significant structural change upon heating to 85 °C or upon addition of significant quantities of chaotropic solvents. To make these peptides more susceptible to denaturation, the p-sheet was extended by two residues. This chain extension was expected to increase the chain entropy, thereby reducing the thermal stability of the peptide. Unexpectedly, the addition of two lysines to the hydrophilic side of gramicidin S (Peptide 5) significandy reduced its p-sheet content under benign conditions. However, the addition of the structureinducing solvent trifluroethanol (TFE) actually enhanced the p-sheet content of this molecule (Figure 3a). While TFE is commonly used to induce helical
455
A Water-Soluble P-Sheet Peptide
Structure in peptides, we believe this represents one of the few instances where TFE has been used to induce the formation of p-sheet structure (18). A
5-1
_-^ o E •a u
0^ i"
•\
o E •o E o
^
''^^^
2.
'o -15H
\
'o
* TFE
B
5
-5
-10 -15
A ',\
5 "C
»\ * ^^
-25 J 190
200
220 230 Wavelength (nm)
240
250|
»C
-----
2! -20
_ 210
. ' • ' • *
' ^^ ^85
^^
X
5;-20
,"' ^ y' /
-25
1
190
200
210 220 230 Wavelength (nm)
240
250|
Figure 3. A) CD spectrum of Peptide 5 with TFE (51% P-sheet) and without TFE (26% Psheet). B) CD spectrum of Peptide 5 at 5 'C and 85 *C. Spectra were analyzed using the program RBOCON (R.F. Boyko, unpubUshed). Another interesting feature of this extended p-sheet model can be seen in Figure 3b, where we show the effect of temperature on the CD spectrum of Peptide 5. Curiously, when the sample temperature is decreased (to 5 °C), the spectrum takes on more of a "random coil" character (only 20% p-sheet); but, when the temperature is increased to 85 X , the spectrum exhibits significantly more psheet character (38% p-sheet). In other words, high temperatures induce structure and cold temperatures reduce structure. We believe that this represents an excellent example of cold denaturation (19), and it suggests that the thermodynamics of p-sheet formation may be more complex than currently appreciated. C. Stabilizing
and Destabilizing
Amino
Acid
Substitutions
To enhance the p-sheet content of Peptide 5, two of its lysines were exchanged for isoleucines. Isoleucine is known to have a stronger p-sheet propensity than lysine (20). However, because these changes were expected to reduce the solubility of the peptide, the two phenylalanines were exchanged for serines. The resulting construct was called Peptide 6. A second construct (Peptide 7) was synthesized wherein one of the isoleucine residues was substituted with a glycine. This substitution was predicted to reduce the p-sheet content of the peptide. In Figure 4 we compare the CD spectra of Peptides 6 and 7. As expected, the spectrum for Peptide 6 has substantially more p-sheet than Peptide 7 (31% p-sheet vs. 2% p-sheet). Indeed, the CD spectrum for Peptide 7 closely resembles that of a classic random coil (21). Furthermore, as judged by the overall shape of the CD curve, the spectrum for Peptide 6 appears to have slightly more p-sheet (31%) than Peptide 5 (26%), as expected. It is also worth noting that Peptide 6, just like Peptide 5, exhibits features of cold denaturation and TFE induced structure stabilization (data not shown). These results suggest that a model based on the sequence of Peptide 6 has many of the features required of an ideal p-sheet model.
Davids. Wishart^r a/.
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Peptide 6
••
200
210
220
230
240
250
1
Wavelength (nm)
Figure 4. CD spectra of Peptide 6 and Peptide 7 collected at 25 'C under benign (aqueous) conditions.
IV. Conclusions This report describes our efforts at designing, synthesizing and characterizing a water-soluble p-sheet analog. We think that we have succeeded in designing a 12 residue cyclic peptide (Peptide 6) which satisfies most of the criteria required of a model p-sheet: it is small (< 20 residues), monomeric, water-soluble, pure (composed of only p-sheets and p-turns), mostly amphipathic, reversibly denaturable, composed of only natural amino acids, relatively easily synthesized and easily characterized by either CD or NMR. We plan to refine this model to enhance its amphipathicity and to improve its cyclization efficiency. Once this refinement stage is complete, we will begin to systematically investigate the influence of amino acid substitutions on both the hydrophilic and hydrophobic sides of this peptide. The resultant data will be used to extract specific p-sheet propensities for all 20 naturally occurring amino acids. In addition to this work on monomeric p-sheets, we are beginning to study dimeric p-sheets (psandwiches) by preparing a variety of disulfide-linked p-sheet analogs. This will allow us to investigate the influences of side chain packing and hydrophobic effects on the stabilization of "idealized" p-sandwiches. We are hopeful that these model systems will provide researchers with the detailed information they need to understand the intricacies of p-sheet and p-turn formation in natural proteins.
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