Anal. Chem. 1995,67, 252-258
Location of an Amphipathic a-Helix in Peptides Using Reversed-Phase HPLC Retention Behavior of D-Amino Acid Analogs Ebellhard Krause,* Michael Beyermann, Margitta Dathe, Wen Rothemund, and Michael Bienert Institute of Molecular Pharmacology, Alfred-Kowalke Strasse 4, 10315 Berlin, Germany
The reversed-phase HPLC retention behavior of amino acid replacement sets of an amphipathic model peptide, neuropeptide Y, and corticotropin releasing factor has been studied. The results demonstrate that D-&O acid substitutions destabitizedthe amphipathic a-helix,leading to a decrease of fractional helicity as determined by circular dichroism. The effect is enhanced by substitution of two adjacent amino acids and correlates well with a decrease of hydrophobic interaction during reversedphase HPLC, caused by disturbance of the preferred binding domain of the stationary phase-bound peptide. In contrast, D-&O acid substitutions in nonamphipathic or disordered regions of peptides do not influence the retention time to the same extent. Thus, the “retention profile”that results from plotting the retention time vs the position of the double a amino acid replacements provides an indication of the presence and location of an amphipathic a-helical secondary structure in peptides. The chromatographic behavior of peptides during reversedphase high-performance liquid chromatography (RP-HPLC) is known to be primarily due to the distinct hydrophobic interactions of the amino acid side chains with the stationary phase. Thus, the chromatographic retention time of a peptide could be correlated with the amino acid comp~sition.’-~Mant et al.4 demonstrated that there is also an effect of the peptide chain length on peptide retention times. Peptides consisting of more than 15-20 amino acid residues tend to be eluted more rapidly than predicted on the basis of the overall hydrophobicity alone. In addition to these two factors, the secondary structure induced by reversedphase interaction may affect the HPLC retention behavior of peptides. The formation of an amphipathic a-helical structure induces a preferred hydrophobic binding domain and leads to considerably stronger retention on reversed-phase c o l ~ m n s . ~ - ~ Biittner et al.8 have investigated single methionine analogs of the model peptide A c - L N H z and have shown (1) Meek, J. L. Proc. Natl. Acad. Sci. USA.1980,77,1632-1636. (2) Sasagawa, T.; Okuyama, T.;Teller, D. C. J. Chromatogr. 1982,240, 329340. (3) Guo, D.; Mant, C. T.; Taneja, A K; Parker, J. M. R; Hodges, R S. J. Chromatogr. 1986,359,449-518. (4) Mant, C. T.; Burke, T. W. L;Black, J. A; Hodges, R S.J. Chromatogr. 1988, 458,193-205. (5) Zhou,N. E.; Mant, C. T.; Hodges, R S. Pept. Res. 1990,3,8-20. (6) Ostresh, J. M.; Biittner, K; Houghten, R k In HPLC of Peptides and Proteins: Separation, Analysis and Confirmation;Hodges, R S., Ed.; CRC Press: Boca Raton, FL, 1991; p 633. (7) Steiner, V.; Sch*, M.; Btimsen, K 0.; Mutter, M. J. Chromatogr. 1991, 586,43-50.
252 Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
that the extent of methionine oxidation is dictated by the accessibility of the Met residues to the aqueous H202 phase according to binding of the peptide to the hydrophobic stationary phase. The results confirm the amphipathic a-helical structure of the peptide bound to the chromatographic phase. Considering the hydrophilic aqueous mobile phase and the hydrophobic stationary phase, the chromatographic system may mimic the anisotropic interface of the cell membrane and can be used to determine the binding sites of proteins by examining sets of overlapping synthetic peptides by reversed-phase HPLCe9 The present study evaluates the relation between the presence of a-helical secondary structure and HPLC retention times. Since incorporation of D-amino acids in a-helical peptide segments is known to decrease the helical content as determined by CD spectroscopy,lOJ1we synthesized D-amino acid replacement sets of the amphipathic a-helical model peptide KLALKLALKAL. KAALKLA-NHz. In order to enhance the effect, two adjacent amino acids of the model peptide were substituted by the corresponding D-amino acid. In addition, a double D-amino acid replacement set of neuropeptide Y (NPY) and of ovine corticotropin releasing factor (oCRF), which have been identified to have an amphipathic a-helicalsecondary structure,were synthesized and studied. NF’Y, a 36residue peptide with an amidated carboxy terminus, was first isolated from porcine brain.I2 The peptide is widely distributed in the central and peripheral nervous system and shows sequence homology to avian pancreatic polypeptide.13J4 The conformation of NPY has been studied by CD, NMR, and molecular dynamics methods. The results indicate a well-defined a-helii comprising approximately 15-18 amino acids of the C-terminus,whereas the N-terminal half of the peptide is di~ordered.’~J~ CRF is the major physiological regulator of the secretion of ACTH and P-endorphin from the anterior pituitary gland17J8and has been shown to (8) Biittner, K; Houghten, R A In Peptides 1990; Giralt, E.,Andreu, D., Eds.; ESCOM Science: Leiden, 1991;p 478. (9) Btittner, IC; Pinilla, C.; Appel, J. R; Houghten, R A J. Chromatogr. 1992, 625, 191-198. (10) Chen, H. C.; Brown, J. H.; Morell, J. L.; Huang, C. M. In Peptides: Chemistry, Structure and Biology; Rider, J . E., Marshall, G. R, Eds.; ESCOM Science: Leiden, 1990; p 122. (11) Pounny, Y.; Y. Shai, Y. Biochemistry 1992,31, 9482-9490. (12) Tatemoto, K; Carlquist, M.; Mutt, V. Nature 1982,296, 659-660. (13) M e n , J.; Novotny, J.; Martin, J.; Heinrich, G. Proc. Natl. Acad. Sci. U.SA. 1987,84,2532-2536. (14) Glover, I.; Haneeff, I.; F’itts, J.; Wood, S.; Moos, D.; Tickle, I.; Blundell, T. Biopolymers 1983,22, 293-304. (15) Mierke, D. F.; Diirr, H.; Kessler, H.; Jung, G. Eur. J. Biochem. 1992,206, 39-48. (16) Darbon, H.; Bemassau, J. M.; Deleuze, C.; Chenu, J.; Rousse, A; Cambillau, C.Eur. J. Biochem. 1992,209, 765-771.
0003-2700/95/0367-0252$9.00/0 0 1995 American Chemical Society
mediate stress-induced changes in the nervous system and in behavior. As a result of structure-activity investigations and CD studies, it was suggested that the CRF binding region is likely to assume an a-helical structure on interacting with its receptor or anisotropic structure^.^^-^^ The effect of the amino acid substitution position on helicity and change of retention time of peptides were correlated in order to locate amphipathic a-helical structures by use of the HPLC retention data for the corresponding double amino acid replacement sets. EXPERIMENTAL SECTION
Chemicals and Reagents. HPLC-grade acetonitrile was obtained from J. T. Baker Phillipsburg, NJ). Water was puri6ed with a Milli-Q system (Millipore, Eschbom, Germany). The eluents were degassed by continuous sparging with helium. All reagents were at least analytical-reagent grade. Trifluoroacetic acid ("FA), phenol, and piperidine were supplied by Merck (Dmstadt, Germany). The Fmoc amino acids and 2-(Wbenzotriazol-l-yl)-l,1,3,3-tetramethyluronium tetrafluoroborate used in synthesis were obtained from Novabiochem (Bad Soden, Germany). Dimethylformamide (DMF) and diisopropylethylamine (DIEA) were obtained from Fluka (Buchs, Switzerland). Triisopropylsilane, 2,2,2-trifluoroethano1 (TFE) , and sinapinic acid were supplied by Aldrich (Steinheim, Germany).
Peptide Synthesis and Fkification.- -
LA-NH2, NPY, oCRF, and the corresponding &amino acid analogs were synthesized automatically (MilliGen 9050 peptide synthesizer) by solid-phase methods using standard Fmoc chemistry in the continuous flow mode VentaGel S RAM resin 0.22 mmol/g (Rapp Polymere, Tubingen), TBTU, 2 equiv of DIEA, coupling 20 min, deblocking with 25%piperidine in DMF for 10 min, final cleavage with 88%TFA/5% phenol/5% H20/2% triisopropylsilane for 3 h) as previously described for the synthesis of CRF analogs.23 Puriiication of 100.mg samples was carried out by preparative HPLC on PolyEncap A300, 10 pm, 250 x 20 mm i.d. (Bischoff Analysentechnik GmbH, Leonberg) to give final products >95% pure by RP-HPLC analysis.24 The peptides were characterized by MALDI-MS, which gave the expected [M HI+ mass peaks and gave correct amino acid analyses. Peptide Characterization. Matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) was performed on a linear timeof-flight mass spectrometerMALDI 11 (Kratos, Manchester) using the positive detection mode and a sinapinic acid matrix. The spectra were obtained by summing over 50 laser pulses (337 nm). The [M HI+ peak of bovine insulin ( m / z 5735) was used for internal mass calibration. Quantitativeamino acid analysis was achieved by ionexchange chromatography and postcolumn derivatization with ninhydrin
+
+
(17) Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Science 1981, 213, 1394-1397. (18) Vale, W.; Rivier, C.; Brown, M. R; Spiess, J.; Rivier, J. Rec. Prog. HOM. Res. 1983, 39, 245-270. (19) Lau, S. H.; Rivier, J.; Vale, W.; Kaiser, E. T.;Kezdy, F. J. Proc. Nutl. Acad. Sci. U.SA. 1983, 80, 7070-7074. (20) Pallai, P. V.; Mabilia, M.; Goodman, M.; Vale, V.; Rivier, J. Proc. Natl. Acud. Sci. U S A . 1983,80,6770-6774. (21) Rivier, J.; Rivier, C.; Galyean, R; Miranda, A.; Miller, C.; Craig, A. G.; Yamamoto, G.; Brown, M.; Vale, W. J. Med. Chem. 1 9 9 3 , 3 6 , 2851-2859. (22) Kaiser, E. T.; Kezdy, F. J. Science 1984, 223, 249-255. (23) Dolling, R; Beyemann, M.; Haenel, J.; Kemchen, F.; b u s e , E.; Franke, P.; Brudel, M.; Bienert, M. J. Chem. Soc., Chem. Commun. 1994,853-854. (24) Krause, E.; Wenschuh, H.; Beyemann, M.; Bienert, M. In Peptides 1992 Schneider, C. H., Eberle, A. N., Eds.; Elsevier Science Publishers: Amsterdam, 1993; p 469.
(Biotronik-Eppendorf LC 3000). Peptides were hydrolyzed in 6 N HC1 at 110 "C for 22 h; norleucine was added for internal standardization.
Circular Dichroism Measurements. CD measurements were carried out on a Jasco 720 spectrometer in TFE/H20 from 185 to 260 nm. The amount of helix was estimated from the relation
where [ 0 ] 2 2 2 is the determined mean residue ellipticity at 222 nm. For [01°222 and [0I1Oo222, representing 0 and 100%helix content, values of -2340 and 30300 degcmz/dmol, respectively, were usedeZ5 For precise determination of peptide concentration, quantitative amino acid analysis was used. Reversed-Phase HPLC. Chromatographic measurements were performed on a Shimadzu LC-1OA gradient HPLC system consisting of two LC-1OAD pumps, a SILlOA autoinjector, a SPDMlOA diode array detector operating at 215 nm, and a CLASS LClO software package. Runs were carried out on a PolyEncap A300 column (250 x 4.6 mm id., 5 pm, Bischoff Analysentechnik GmbH, Leonberg). The sample concentration was 1 mg/mL of peptide in 0.1% TFA (eluent A) with an injection volume of 20 pL. Separations were performed at 22 "C (thermostated system) and at an eluent flow rate of 1 mL/min. The precision of the retention times was f0.05 min. Mobile phase A was 0.1%TFA in water and B was 0.1%TFA in 50% acetonihile/50% water(v/v) for KLALKLALKALKAALKLAamide and NPY, and A was 0.1%TFA in water and B was 0.1% TFA in 70%acetonitrile/30%water (v/v) for CRH. The retention times of KLALKLALKALKMLIUA-amide peptides were determined using a linear gradient 5-95% B in 40 min, whereas NPY peptides were analyzed using a linear gradient 40-95% B in 40 min and the CRH peptides with a linear gradient 20-80% B in 40 min. RESULTS AND DISCUSSION
Effect of a amino Acid Substitutions on Helicity and HPLC Retention Behavior of P N H2. The 18mer model peptide used was derived from Mutter's template-assembled synthetic proteins (TASP)approach? in which secondary structure-forming peptides are attached to a template. The amino acid sequence of the peptide with a high potential to form an amphipathic a-helical structure consisted exclusively of leucine, lysine, and alanine residues and is illustrated as the helical wheelz6of Figure 1. In order to innuence the stability of the helical peptide, two sets of peptides were synthesized including 9 and 18 analogs of -amide, respectively. The analogs d ~ e r e din the successive replacement of each amino acid position with its Denantiomer (D-replaCement)and in the successive replacement of two adjacent amino acids with the corresponding D-aminO acid (D~replacement) . The a-helicity of the peptides synthesized was determined by CD as shown in Figure 2. Although the precise mechanism of (25) Chen, Y. H.; Yang, J. T.; Martinez, H. M. Biochemistry 1 9 7 2 , 11, 41204131. (26) Schiffer, M.; Edmundson, A. B. Biophys.]. 1 9 6 7 , 7, 121.
Analytical Chemisfty, Vol. 67, No. 2,January 75,7995
253
Table I. Fractional Hellclty and RP-HPLC Retention Times* of D-Amino Acid Analogs of KLALKLALKALKAALKLA-amide D-aminO
acid retention substitution time (min) helicity
peptide sequenceb KLALKLALKALKAALKLA KLALKLAL-
Figure 1. Helical wheel projectionz6of KLALKLALKALKAALKLAamide. 1
8
-
o,8h k\
-2
-3 -4 -5
-6
29.2 32.2 30.5 31.1 28.4 28.7 27.9 28.3 27.8 26.4 26.6 25.7 27.3 26.5 26.9 27.2 30.1 29.4 29.2 30.9 28.6 26.7 26.2 23.5 23.4 24.4 25.8 29.8
K' L2 A3 L4 K5 LE A7 L8
K9 A10 L" K'2 AI3 A14 L'5 K'6 L'7 A18 K1,L2 A3,L4 K5,L6 A7,L8
[degcm2/dmol] -rsll-i:peptlde
none
I
K l L2 A? L8 K9 A10 L11 K12 L15 K16
0.6
0.4
PA10
L",Kl2 A13A14 L15,K16 L17A18
(%)
54 63 55 56 53 52 44 42 49 43 48 32 48 46 42 44 52 48 51 60 45 38 34 25 22 26 40 49
a The retention times were determined on PolyEncap MOO. The one-letter code of the Damino acids is underlined.
helicity (%,,) 185
200
215
230
A[nml
260
Figure 2. CD spectra of KLALKLALKALKAALKLA-amideand some of its analogs with D-amino acid substitutions in 50% TFE/50% 0.01 M HsP04 (v/v), 5 x mol/L.
--I
701
60structure induction is unknown, 2,2,2-trinuoroethanol (TFE) can induce the formation of helical structures in peptides which are unstructured in aqueous s o l ~ t i o n . 2CD ~ ~measurements ~~ in water and water/TFE mixtures (TFE titration) indicated that maximum helicity of the KLALKLALKALKAALKLA-amide occurs between 50 and 90%TFE, and therefore a solvent system containiig 50% TFE was used in CD studies. The helicity data and the RP-HPLC retention times of all -amdie analogs are summarized in Table 1. The plot of retention time vs helicity (Figure 3) clearly demonstrates that differences in RF-HPLC retention behavior caused by D-amino acid substitutions correlate well with the helicity (R = 0.92). Inversion of the amino acid conliguration from position 4 to 18 of the amphiphathic a-helical model peptide leads to a significant drop of the helicity (Figure 4.4). The effect is stronger in the center of the helix and is enhanced by substitution of two adjacent amino acids, as shown in Figure 4B. A similar shape of a helix-destabilizing effect was described by Baldwin et aLB for a series of single alanine-glycine (27) Sannichsen, F. D.; Van Eyk, J. E.; Hodges, R S.; Sykes, B. D. Biochemistry 1992,31,8790-8798. (28) Jasanoff, A; Fersht, A R Biochemistry 1994,33,2129-2135. (29) Chakrabartty, A; Schellmann, J. A; Baldwin, R L. Nature 1991,351,586588.
254 Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
R = 0.92
0 20
25
30
35
retention time (min) Figure 3. Correlation of reversed-phase HPLC retention times of KLALKLALKALKAALKLA-amide(+) and 27 o-amino acid analogs with the CD-determined helicities measured in 50% TFE/50% water (v/v).
substitutions in an alaninerich model helix. The difference in the helix propensity of alanine and glycine led to the largest drop in CD-determined helix content in the center of the helix, and substitutions close to either end have comparatively small effects.
-30
-30 -
-40
TFEhO% 0.01 M H3P04 (v/v), 5 x
N
2-
t
4-
W 2-
(D
z
$!
z
:
'D
moi/L. (A) D-Replacement; (B) DD-replacement.
B
A A
A retention time (min)
retention time (min)
2
2
0
0
.2 -2
-4 -4
+W +Q : : z : z
2 ; 2 2 $ " ; $ $ N
~ y ~ s ~ 3 ~ a5 A9 ~P az a9 a2 ~s A: a~ s s position substituted
e
y
i
Z
Z
position substituted
Figure 5. Change in retention time of KLALKLALKALKAALKLA-amide vs position of D-amino acid substitution Experimental Section for chromatographic details. (A) D - R e p h " t ; (B) DD-replacement.
Figure 5 shows the deviation of HPLC retention times of the model peptide analogs caused by D-amino acid substitutions (A) and double a amino acid substitutions @). The results indicate similar effects on retention time and helicity. Although such substitutions should not influence the overall physicochemical properties of the peptide, replacement of amino acid residues in the helical region of the molecule by their Disomers leads to a decrease of the retention time of analogs in reversed-phase chromatography. This change in retention behavior may be related to the ability of the peptide to form a helix on binding. In the case of an amphipathic a-helix,the molecule forms a preferred
i
peptide,
29.2 min). See
hydrophobic binding domain at one site of the helix, as described by Hodges et al.5 @Amino acid residues incorporated in the helical area of a peptide may make it more difficult to form a helical structure and thereby the organization of a hydrophobic binding domain, thus reducing the retention time on reversed-phase stationary phases. The introduction of two amino acids intensifies the effect on both helicity and retention time. Replacement of amino acids in positions 1-3 of the N-terminal region of the peptide with their D-enantiOmerS did not signzcantly influence the molar ellipticity nor the retention time, indicating that the helical content in this part of the peptide chain is extremely weak. Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
255
Table 2. Fractional HelGity and RP-HPLC RetentSon Times* of D-Amino Acid Analogs of Neuropeptide Y Damin0
peptide sequenceb
acid substitution
retention time (min)
helicity
(%)
67 19.9 YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY 71 20.0 YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY 67 20.1 FSKPDNPGEDAPAED~L~RYYSALRHYINLITRQRY 67 19.9 YPSKPDNPGEDAPAEDMNLITRQRY 62 19.7 YPSKPWGEDAPAED~URYYSALRHYINLITRQRY 63 19.0 YPSKPD WGEDAPAEDMNUTRQRY 7u 20.5 YPSKPDNPGEDAF%~EDMARYYSALRHYINLITRQRY 55 18.2 YPSKPDNPGEDAPAEDMARYYSALRHYINLJTRQRY 56 18.3 YPSKPDNPGEDATTED~~ARYYNRHYINUTRQRY 44 16.8 Y R Q RTU N A DEGPN DPKSPY 49 14.7 YPSKPDNPGEDAPAEDRXRYYSALRHYINUTRQRY 53 16.8 YPSKPDNPGEDAPAED-NLITRQRY 54 12.0 YPSKPDNPGEDAPAEDMARWFALXHYINLITRQRY 56 13.7 YPSKPDNPGEDA 41 14.1 YPSKPDNPGEDA 53 16.5 YPSKPDNPGEDAPAEDMARYY~TRQRY 38 15.9 YPSKPDNPGEDAPAEDM"RQRY 58 21.4 YPSKPDNPGEDAPAEDMARYYWJWINUTTRQRY 66 20.3 YPSKPDNPGEDAPAEDMARYYSALRHYINLITR~ @
The retention times were determined on PolyEncap MOO. b The oneletter code of the Damino acids is underlined.
This result clearly demonstrates that the differences in RPHPLC retention behavior caused by mreplacement correlate well with the helicity. Thus, the characteristic retention time profile (Figure 5 ) fits with the corresponding plot of the CD data and should allow the location of amphipathic helical structures in native peptides. Helicity and Retention Behavior of a DPReplacement Set of Neuropeptide Y. Considering that NPY may form a helical structure on reversed-phase columns, a double Damino acid replacement set was synthesized in order to evaluate the relation between helicity and HPLC retention time. Since in NPY a substantial amount of helix can be induced in the presence of TFE,I5CD spectra of NPY analogs were measured in 50%TFE/ 50%HzO. The fractional helicity data and the RP-HPLC retention times of NPY and of the Dbreplacement set are summarized in Table 2. The influence of the position of &amino acid substitution of the a-helicity of NPY analogs, as demonstrated in Figure 6, shows that (i) replacements in the N-terminal region (position 1-11) did not significantly influence the helical content and (ii) the helicity of D-amino acid analogs with substitutionsin the assumed helical region between positions 13 and 34 is reduced compared to that of the all-L peptide. The dependence of the RP-HPLC retention time on the position of substitution, as shown in Figure 7, demonstrates that D-amino acid analogs having substitutions in the helical area of NPY are consistently retained for shorter times than NPY itself and its N-terminal DD-dn0 acid analogs. A similar effect was recently described for single-point D-amino acid-substituted NPY analogs which were synthesized to allow study of their YI and Yz receptor binding aff11ities.3~3~~ However, the effect of the single D-amino acid substitution on the retention time was less pronounced and could not be correlated with CD data. Using the DD-replacement, it is obvious that the iduence (30) Kirby, D. A; Boublik, J. H.; Rivier, J. E. J. Med. Chem. 1993,36,38023808. (31) &ilar, M. I.; Mougos, S.; Boublik, J.; Rivier, J.; H e m , M. T.W. J. Chromatogr. 1993,646, 53-65.
256
Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
A helicity (%,,)
-151
-20 -
-25
-30
position substituted Figure 6. Influence of the position of D-amino acid substitution on the helicity of NPY (helicityatl'LNPY, 67%). 50% TFE/50% H20 (v/v), pH 3.5,5 x mol/L.
of the amino acid substitution position on helicity correlates with change of peptide retention data. The quite similar profiles indicate a destabilization of the secondary structure caused by double D-aminO acid substitution in positions 13-32 of NPY and therefore confirm an amphipathic a-helical domain in this peptide region, as previously suggested by Allen et al.13 The finding that the C-terminal amino acids 34-36 are not involved in the helix agrees with the crystal structure of the homologous avian pancreatic p01ypeptide.l~Furthermore, the retention time profile of the Dbreplacement set suggests a beginning of the helix before
Table 3. RPmHPLC Retention Timesa of o-Amino Acid Analogs of oCRF
Damin0
acid substitution
peptide sequence SQEPPISLDLTFHLLREVLEMTKADQLAQQAHSN~IA
none
SQEPPISLDLTFHLLREVLEMTKADQLAQQAHSNRKLLDIA ~PPISLDLTFHLLREVLEMTKADQLAQQAHSNRKLLDIA
S1Q2 E3P
retention time (min)
24.1 24.2 24.2 23.7 23.0 23.1 23.2 21.5 21.2 19.7 21.7 SQEPPISLDLTFHLLREVLEMTKADQLAQQAHSNRKLLDIA 23.4 SQEPPISLDL 24.2 D25Q26 23.9 L27A28 23.8 23.7 24.0 24.3 24.1 SQEPPISLDLTFHLLREVLEMTKADQLAQQAH-IA R35P6 L37L38 24.2 D39I40 24.0 SQEPPISLDLTFHLLREVLEMTKADQLAQ~AHSNI40A41 24.1
a
The retention times were determined on PolyEncap A300. The one-letter code of the d amino acids is underlined.
position 15, which supports previous NMR studies of NPY published by Darbon et al.16 However, in the present study, no indication for the presence of a hinge inducing a 100" angle between two helical domains (13-26 and 28-35) has been found by the HPLC method. Location of the Amphipathic a-Helical Structure in Corticotropin ReleasingFactor. The positions of the Damino acid substitutions and the retention times of the 22 oCRF analogs synthesized are listed in Table 3. Because of the effective shielding of the silanophilic interactions and the high efficiency for its separation of CRF peptides,24a polymercoated large pore silica material was used as the stationary phase. The influence of the position of pamino acid substitution on the retention time of CRF is demonstrated in Figure 8. The "retention profile" that results from the deviation of the retention time vs position of the mreplacement shows a drastic decrease of retention times from position 7 to 20, suggesting the center of the amphipathic part of the helix comprising residues 13 to 20. In marked contrast, double D-amino acid substitutions in the less amphipathic region (2336) and in the predominantly disordered C-terminal region of CRF did not influence the retention times to the same extent. Recently published structure-activity studies involving single-point D substituted CRF analogs indicated a similar but less pronounced reduction of retention times between positions 8 and 22.2l Although the mechanism of structure induction may be different on FG-HPLC and in TFE/water, the location of the amphipathic a-helix using the retention profile of the m-replacement set correlates well with previous NMR results obtained for CW in 66%TFE/34% suggesting an amphipathica-helix between positions 6 and 20. Unequivocally, the retention data show an extended amphipathic a-helix from residue 7 to residue 22. An amphipathic helix in the Gterminal region (residues 36-41), as suggested by prediction methods,2O could not be confirmed.
A
retention time (min)
0
-2
-4
-6
-8
I
position substituted Figure 7. Change in retention time vs position of DD-replacement - ~ 19.9 min). See Experimental Section for chroin NPY ( t ~ " I l NPY, matographic details.
Although CRF is more than 70% h e l i ~ a P under ~ * ~ ~ structureinducing conditions (>60%TFE), which is in agreement with (32) Romier, C.; Bemassau, J. M.; Cambillau, C.; Darbon,H. Protein Eng. 1993, 6, 149-156. (33) Dathe, M.; Zinver, D.; Gast, K; Beyermann, M.; Krause, E.; Bienert, M. In PEPTIDES Chemistry, Structure and Biologx Hodges, R S., Smith, J. A, Eds.; ESCOM Science: Leiden, 1994; p 544.
Analytical Chemistry, Vol, 67, No. 2, January 15, 1995
257
A retention time (min)
P-
position substituted Figure 8. Change in retention time vs position of DD-replacement in oCRF (tR"'-L CRF, 24.1 min). See Experimental Section for chromatographic details.
statistical analysis methods, the double Pamino acid substitutions in the suggested hydrophilic a-helix (23-26)32 did not significantly iduence the retention behavior on reversed-phase stationary phases. That might be explainable by either a lack of helix formation in this region in the presence of a hydrophobic environment or a less efficient helix disturbance by double D-amino acid replacement.
258 Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
CONCLUSION
On examining the effect of secondary structure on HPLC retention times, we clearly demonstrated that destabilization of the amphipathic a-helical domain in peptides occurs upon a amino acid substitution. The reduction of the fractional helicity as determined from CD spectra correlates well with a decrease of hydrophobic binding to reversed-phase stationary phases. The effect is caused by disturbance of the preferred binding domain of the peptide. Plotting the retention times vs the position of D-amino acid substitution of a replacement set gives a characteristic pattern, showing decreased retention times in the helical region. This "retention profile" is enhanced by Dpreplacements, i.e., replacement of adjacent amino acids in pairs, and provides an indication of the presence and location of an amphipathic a-helical structure in peptides. The HPLC method can be used to assist conformationalanalysis, since small quantities of a large number of analogs are quickly available by multiple techniques of peptide synthesis and do not require excessive purification of the peptide analogs, as is necessary when using CD measurements. Work is continuing in order to examine the importance of the amphipathicity as well as the simultaneous presence of different structural elements in one peptide on the HPLC retention behavior. ACKNOWLEDGMENT
This research was supported in part by Grant Kr 1451/2-1 from the Deutsche Forschungsgemeinschaft. The authors thank Annerose Klose, Dagmar Smettan, Barbara F'isarz, and Heike Nikolenko for technical assistance. Received for review May 11, 1994. Accepted October 26, 1994.Q AC940464D e Abstract published in Advance
ACS Abstracts, December 1, 1994.
