J . A m . Chem. SOC.1986, 108, 4631-4642
4631
Conformations of Cyclic Octapeptides. 3. cycle-( D-Ala-Gly-Pro-Phe)2. Conformations in Crystals and a T I , Examination of Internal Mobility in Solution Kenneth D. Kopple,*+ Krishna K. Bhandary,f Gopinath Kartha,tss Yu-Sen Wang," and Kumarapuram N. Parameswaranll Contribution from the Department of Chemistry, Illinois Institute of Technology, Chicago, Illinois 60616, and Biophysics Department, Roswell Park Memorial Institute, Buffalo, New York 14263. Received September 30, 1985
Abstract: Two crystalline forms of c y c b ( ~ - A l a - G l y - ~ - P r o - ~ - P hcontaining e)~, different peptide backbone conformations, . I, have been subjected to X-ray crystallographic analysis. Both forms are in the orthorhombic space group P 2 1 2 1 2 1 Form with dimensions a = 28.412 (2), b = 17.036 (2), and c = 8.959 (1) A, contains one octapeptide molecule and two water molecules in the asymmetric unit. Its peptide backbone has only trans peptide bonds and approximate C2 symmetry, with type I @ turns at L-Pro-r-Phe. Except for the type I turns, this backbone is similar to that reported for cyclo-(D-Ala-Gly-L-Pro-D-Phe)2, which has type I1 turns a t L-Pro-D-Phe. Form I1 has dimensions a = 32.166 (2), b = 16.896 (2), and c = 8.156 ( I ) A. Its asymmetric unit contains one octapeptide and six water molecules. The peptide backbone of form I1 also contains only trans peptide bonds and type I @ turns at L-Pro-L-Phe but differs from that of form I in that the plane of one peptide bond joining Ala to Gly is rotated by about 180' relative to the local plane of a-carbons. Measurements of proton spin-lattice relaxation in the rotating frame, TI,, were made on dimethyl sulfoxide solutions of cyclo-(D-Ala-Gly-L-Pro-L-Phe)z, a peptide that is indicated to be conformationally mobile in solution by the ambiguity of the usual solution N M R observations. These were compared with similar measurements of cyclo-(D-Ala-Gly-~-Pro-~-Phe)~, which previously reported studies indicated to have a more stable backbone conformation. Significantly shorter values of TI, for the former peptide represent contributions to relaxation of its protons by intramolecular motions in the 104-105-s-' range.
Four diastereomeric cyclic octapeptides, c y c l o - ( ~ -o r L-Ala~, in a search for new classes of Gly-L-Pro+- or ~ - P h e ) prepared conformationally stable cyclic peptide backbones, have been under study. Two of these, cyclo-(~-Ala-Gly-~-Pro-~-Phe)~ and cyclo-(r-Ala-Gly-~-Pro-~-Phe)~, were judged by N M R criteria t o have conformationally stable all-trans backbones, and conformations were proposed for them on t h e basis of N M R measurements of dimethyl sulfoxide so1utions.l A crystal structure of t h e D-Ala,D-Phe peptide, showing a backbone conformation similar to a likely solution conformation, has been reported.2 The L- Ala-L-Phe isomer was proposed t o have a backbone similar to that of @-amanitin3and a n amanitin a n a l o g ~ e . ~ I n this paper we describe solution N M R studies and X-ray crystallographic analyses of the D-Ala,L-Phe diastereomer, cyc/o-(D-Ala-Gly-LPro-~-Phe)~. T h e all-trans form of cyclo-(~-Ala-Gly-~-Pro-~-Phe),, which occurs to the extent of 90%in dimethyl sulfoxide solution, appears to possess a greater degree of conformational mobility than the D-Ala,D-Phe isomer. The indications a r e ambiguity of the usual solution N M R observations and the observation, reported here, of two different conformations in t h e crystalline state for the D-Ala,L-Phe peptide. I t was therefore worthwhile to compare the two isomers with regard t o proton spin-lattice relaxation in t h e rotating frame, T I , ,to determine if the increased mobility occurs on the time scale of motions particularly affecting T I , , 104-10-5 s in our studies. Faster T I ,relaxation was indeed observed for the D-Ala,L-Phe peptide. These crystallographic and NMR results a r e presented below.
Experimental Procedures The synthesis and characterization of cyclo-(D-Ala-Gly-L-Pro-L-Phe)2 were described earlier.' X-ray Analyses. Crystals of form I were grown by slow evaporation of a solution of the octapeptide in methanol-water. Form I1 was obtained by slow evaporation of a methanol-water solution containing sodium thiocyanate. Both forms crystallize in the orthorhombic space group P2,2,2, with unit cell dimensions u = 28.412 (2), b = 17.036 (2), and Illinois Institute of Technology. Present address: Smith Kline & French Laboratories, Philadelphia, PA 19101. * Roswell Park Memorial Institute. *Deceased 1984. 11 Illinois Institute of Technology.
0002-7863/86/1508-4637$0l.50/0
c = 8.959 (1) 8, for form I and u = 32.166 (2), b = 16.896 (2), and c
= 8.156 (1) 8, for form 11. Both forms contain 4 formula units, with a calculated density of 1.196 g/cm3 for I and 1.278 g/cm3 for 11. Intensity data to 28 = 154' for both forms were measured with an Enraf-Nonius CAD-4 automatic diffractometer using Cu Ka ( A = 1.5418 A) radiation. A crystal of size 0.3 X 0.2 X 0.4 mm, mounted in a capillary with mother liquor, was used to collect a total of 4989 independent reflections from form 1. A crystal of form 11, 0.3 X 0.4 X 0.5 mm, mounted on a glass fiber, was used to measure 5256 independent reflections. Intensity data were corrected for Lorentz-polarization effects, and an empirical absorption correction5 was also applied. Structures of both forms were solved by using the direct methods program MULTAN 806 and successive weighted Fourier maps. The asymmetric unit of form I contains one octapeptide and two water molecules for a total of 56 non-hydrogen atoms. That of form I1 contains one octapeptide and six water molecules, 60 non-hydrogen atoms. The structure of form I was refined by the block-diagonal least-squares technique using 3145 reflections with Fo > 1 . 5 aF. The atoms were treated isotropically in the initial stages and with anisotropic thermal parameters in the final stages of refinement. Hydrogen atom positions were calculated and included in the refinement in the final stages. The final R factor was 0.12 for 3145 reflections. The refinement and distance and angle calculations for form I were done by using local programs. Form I1 was refined by the full-matrix least-squares technique using 3898 reflections with I > 2aI. In the initial stages of refinement all the atoms were treated isotropically. Anisotropic thermal parameters were used in the final stages for all the atoms except OW6, which has very high isotropic temperature factor. Hydrogen atom positions were located from a difference map and were included in the calculation but not refined. The final R factor for form I1 was 0.058 for 3898 reflections. The final shift/error was 0.60, and the goodness-of-fit parameter was (1) Kopple, K. D.; Parameswaran, K. N.; Yonan, J. P. J . Am. Chem. SOC.
1984. 106. 7212-7217.
(2) Kopple, K. D.; Kartha, G.; Bhandary, K . K.; Romanowska, K. J . Am. Chem. SOC.1985, 107, 4893-4897. (3) Kostansek, E. C.; Lipscomb, W. N.; Yocum, R. R.; Thiessen, W. E. Biochemistry 1978, 17, 3790-3975. (4) Shoham, G.; Rees, D. C.; Lipscomb, W. N.; Zanotti, G.; Wieland, Th. J . Am. Chem. SOC.1984, 106, 4606-4615. (5) North, A. C. T.; Phillips, D. C.; Matthews, F. C. Acta Crystallogr., Sect. A: Cryst. Phys., D i f j , Theor. Gen. Crysrallogr. 1968, A24, 351-359. (6) Main, P.; Hull, S . E.; Lessinger, L.; Germain, G.; DeClarq, J.-P.; Woolfson, M. M. MULTAN 80, A System of Computer Programs for the Automatic Solution of Crystal Structures from X-ray Diffraction Data; Universities of York and Louvain: York, England, and Louvain, Belgium, 1980.
0 1986 American Chemical Society
Kopple et al.
4638 J . Am. Chem. SOC.,Vol. 108, No. 15, 1986 L-Pro C8cc8‘2\
I
Glv
D-Ala
C8b2
i””
C861-
F3b-c3v,
/“
-c2a
\
L-Phe
nv
c46
\
D-Ala
L-Pro
I
L-Pho
04
GlY
Figure 1. Numbering scheme used for the cyclic octapeptides.
Figure 2. Stereoview of cyclo-(~-Ala-Gly-~-Pro-~-Phe)~, form I. aCarbons are numbered.
1.42. The calculations for form I1 were carried by using the Structural Data Package. The final atomic coordinates and structure factor tables for both the forms are available as supplementary material. NMR Measurements. IH NMR data were obtained by using a Nicolet NT-300 spectrometer, operating at 300 MHz for protons and 75 MHz for carbon. Peptide concentrations were 10 mM or leas for proton spectra and 10-25 mM for carbon spectra. Proton TI, measurements were made at 20° on 7 mM degassed sealed samples in 5-mm tubes. Excitation and spin-locking power were obtained from the low-power transmitter of the spectrometer augmented by an EN1 IO-W linear amplifier. The usual 90,-r(SL,) acquisition sequence was used. A pair of crossed diodes at the amplifier output reduced noise during acquisition. To ensure that accurate T I Pvalues were obtained, the protons examined had resonances free of overlaps, and separate experiments, with the locking field, H I ,at the Larmor frequency of each proton, were made for each proton. Relaxation measurements were made at five or six different field strengths between y(Hl) = 2600 and 8600 Hz, using at least 10 spin-locking times between 6 ms and 2 s. Values of T I Pwere obtained by fitting resonance intensities to the expression I = Io exp(-rst/Tlp) + I,, where I, represents a residual signal present at long spin-locking times (>lOTI,), usually
