Methylproline; NMR; fl-Turn; Conformation; Bradykinin. 1. INTRODUCTION. Proline residues frequently occur in reverse turns on the surface of globular proteins ...
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© 1992 Federation of European Biochemical Societies 00145793/92/$S.00
fl-Turns induced in bradykinin by (S)98% pure by reverse-phase HPLC, and gave satisfactory amino acid analyses, FAB mass spectra (glycerol/thioglycerol/a~otic acid matrix), and tH, '~C and JSN NMR spectra. 2.2. NMR spectroscopy Two dimensional NMR experiments wore carried out on a Bruker AMX-600 in 90% H20-10% D20 at pH 4. Proton chemical shifts are relative to external TSP. All spectra were recorded in phase-sensitive mode with quadrature detection in the FI dimension using timeproportional phase incrementation [9]. kH spectra typically had a spectral width of 5555 Hz (9.25 ppm) in F2 and were collected with 2048 time domain data points. In FI 512 h-increments w¢ro recorded to yield a size of 1024 × 2048 for the final (real) 2D matrix after zero-filling in each dimension. All data wean enhanced in both directions using cosine bell window functiona, naselin,~ flattening was carried out using an automatic third order polynomial baseline correction routine in both dimun~ions. TOCSY [10,11] spectra wean per.
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Ibnned with a 200 ms MLEVI7 spinlock (TB.,/2g ~ 7500 Hz) surrounded by two 2.5 ms purging pulses to remove phase distortions. ROESY Spectra [12] were recorded with 100 ms and 300 ms spinloek times (TB2/2~r= 3600 Hz). Water suppressions for these experiments was achieved with low power pre-irradiation. 3. R E S U L T S
3.1. Assignment procedure, chemical shifts and coupling constan Is T h e strategy used to assign p r o t o n s p e c t r a [13] relied u p o n the identification o f spin systems f o r individual a m i n o acid residues in D Q - C O S Y [14] a n d T O C S Y spectra [10,11], followed by d e t e r m i n a t i o n o f neighb o u r i n g a m i n o acids f r o m the o b s e r v a t i o n o f sequential N O E connectivities. T h e tH a s s i g n m e n t s are given in T a b l e I. R e s o n a n c e s f r o m the segment Pro~-Glya-Phe % Ser 6 were identified readily b y an N O E walk t h r o u g h the C = H - N H conncctivities, starting f r o m the G l y 4 a m i d e triplet. T h e o n l y m i n o r difference between the a s s i g n m e n t s d e d u c e d here for B K at p H 4 (not reported), a n d t h o s e o b t a i n e d earlier at p H 7.4 in D.,O arise in A r g t [7]. F o r example, at the lower p H the A r g ~ C=H a p p e a r s 0.23 p p m to l o w e r field [15]. All the o b s e r v e d 3J~r~rt values are close to the timea v e r a g e d value o f 6.3 H z f o r free r o t a t i o n a b o u t the
N-C,~ b o n d [16], and as the expected values f o r residues at positions i+ i and i+2 in fl-turns in proteins also lie in the range 4--9 H z [13], n o firm conclusions a b o u t preferred turn c o n f o r m a t i o n s can be d r a w n f r o m these data. Also, the 3J~a c o u p l i n g c o n s t a n t s observed f o r the a n a l o g u e s (Table 1) are m a i n l y consistent with rapid a v e r a g i n g between t w o or m o r e distinct values o f Xj. O n l y the side chain o f Phe a in 3 - ~ M e P r o - B K a n d 7g M e P r o - B K has '~J,¢ c o u p l i n g c o n s t a n t s which suggest that o n e o f the g a u c h e r o t a m e r s or the trans r o t a m e r is f a v o u r e d [16,17]. A t present the f a v o u r e d 2', angle c a n n o t be identified because the fl p r o t o n s have n o t been stereospecifically assigned. 3.2. Temperature dependence o f NH-chemical shifts A n a m i d e p r o t o n involved in a stable i n t r a m o l e c u l a r h y d r o g e n b o n d , or o n e inaccessible t o solvent for steric reasons, typically s h o w s a reduced t e m p e r a t u r e coefficient in the range 0 t o - 3 x 10 -3 p p m / K [18]. T h e a m i d e t e m p e r a t u r e coefficients for BK, 3-txMePro-BK a n d 7g M e P r o - B K are s h o w n in T a b l e !I. O n l y the A r g 9 N H shows a r e d u c e d t e m p e r a t u r e coefficient, which is c o n sistent with its involvement in a h y d r o g e n b o n d e d t u r n c o n f o r m a t i o n . H o w e v e r , b o t h the cis and trans rotamers at S e r - P r o - P h e in B K show a reduced t e m p e r a -
Table I tH-Chcmical shift assignments (8 ppm) and ~d~ (Hz) coupling constants for the major trans rotamgrs of the peptidcs RI'P~'GFSPFR and RPPGFSP~°FR in 10% D:O/H,O at pH 4.0 and 286 K (P~ = (S).a-methylproline) R
p
pr~l,
G
F
S
P
F
R
.... ....
8.02 3.69
7.83 4.37
8.00 4.49
.... 4.12
7.91 4.44
7.61 3.96
1.99 1.82
....
2.89
3.57
1.96
. . . . . . . . . . . .
1.99 1.50 1.70
3.03 2.79 ....
1.64 1.52 1.34
1.91 3.77
. . . . . . . . . . . .
3.39
....
2.99
9. I
7.02 7.9
4.4
6.0
5.2
S
p~t~
F
R
8.18 4.60
----1.39 1.70 i .38 1.82 1.59 3.57 3.52 . . . ....
Na.[- ~
. . . . . . . .
C=-H r~Me C:-H
4.14
4.56
1.68
C:H
1.47
2.21 1,69 1.83
C:H N,-H ~J~ (Hz)
1.37
2.86 7.00 6.1
R
N,-H Ca-H a-Me C#-H Cr-H C6-H N:H 3j~ (Hz)
. . . . . . 4.33 . . . . . 1.87 .... 1.68 . . . . . 3.10 .... 7.16 5.9
3.52 3.26 .... nd
3.56
. . . . . . . . . . . . . . . . . . . . . . . ........ 7.4 6.7 8.5
P
. . . . . 4.76 . . . . 2.42 1.87 2.00 . . . . 3.74 3.47 . . . 7.5
P
.
G
8.37 4.41 3.87 . . . . . . . . . . 2.28 .... 1.89 ........ 2.03 . . . . . . . . . . . . . . . . . 3.81 . . . . 3.65 . . . . . . . . . . . . . . . 8.2 .... 5.3
F
7.93 4.56 . . . . 3.03
3.76 3.67
. . . . . . . . . . . . . . . . . . . . . . . . . . 7.0 7.0
.
.
7.49 4.65 ........ 3.29 2.84 --........ --........ 10.0 4.7
7.68 4.13 1.82 1.7 I 1.57 3.16 7.15 7.6 5.9 217
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R P P G F S P F R
R P P B F S X F R i . . . . . . . .
I.-
B.-J.
-.-
95% the all-trans configuration. The ~methylproline at position 7 is expected on steric grounds to strongly favour the trans-Ser6-MePro 7 rotamer, Apart form the normal ROE connectivities between protons within one residue, or in adjacent residues, a number of medium range gOEs were again seen which indicate that turn conformations are stabilised (relative to BK) in both the Pro~--Phes and Se:-Arg '~ segments of this peptid¢, Observed ROE connectivities that are diagnostic for a reverse turn at Se:-Arg ° include Arg o N H to S e : C:H, Are 9 to either ProT-C~Me or Pro%C/~H (coincident resonances), Are '~N H to Phe a NH, and Phe s N H to Set 6 C/jH (Fig. 2). The ROEs observed between Arg o and Se:, as well as Are 9 and Pro 7 confirm the presence of a reverse turn formed by residues Ser%ArgL Similar evidence for a turn conformation at Pro-'-Phe s includes ROEs observed between Phe s N H and Pro ~ C~H, between Phe s C#H and Pro" C6H, and between Phe ~ N H and Gly 4 NH. Thus the expected stabilisation by a-MePro of a turn conformer at Se:-Arg 9 in this case is accompanied by an additional effect at Pro"Phe s. 3.4. Biological activity The biological activity of the analogues is under investigation and the results will be reported in detail elsewhere. 4. DISCUSSION In agreement with earlier studies, the R O E data reported here suggest that BK does not adopt a stable secondary structure on the N M R timescale in aqueous
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A
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B O Pro3M¢.Ph,'SN
-2.8
-1.4
•
Arg9Ng'
~
ArggNB"
\
"3.0
-1.6 -3.2
Pro2g-PheSN
__:-i .8
0
-3.4 ArggN-Ser6B
D~
-=ppm pprn
7.7
7.15
ppm
7.7
-ppm
7.13
D
C "i .4 Arg9N.ProTMg
~'ArggN-Phe@B'
Phe@N-Pro7Me
a~ /
-3.0
Arlt9N-PhegB"
~a
PhQgN.Ser6~':"X ~!~ ~''" O0.
p~m :llltit[lllttllll]Zl ppm 7.6
[111ii111]I~,
I
7.5
-3.5
PhegN-$¢r6g'~
-i .8
tll,ll~i~ PDm
Arg9N Ser6B'N~ Arg9N-Ser6fl"
ppm
ii,[l~aJ~,~Jlrt,,tlt 7.6
7.5
Fig. 2. Portions of 600 MHz phase sensitive ROESY spectra: A and B from 3.aMePro.BK at 286 K; C and D from 7-aMePro-BK at 300 K. Both in 90% H,O-10% D,O, oH 4. ROEs from neighbouring residues, as well as longer range effects are indicated. The mixing time for both was 300 ms.
solution [7,19]. However, the 3-gMePro-BK and 7czMePro-BK analogues show clear evidence for the increased population of reverse-turn conformations in 90% H20-10% DuO. In each peptide, ROE connectivities indicate the presence of two families of turn conformations, one centred on the residues Pro2-Phe ~ and the other at SeP-Arg°. However, the ROE connectivities do not allow a distinction to be drawn between type-I or type-lI turns, nor do they indicate whether both turns are populated simultaneously. Both the ProL ProLGly4-phe 5 and SeP-ProT-PheLArg9 tetrapeptide sequences have a high predicted propensity tbr forming fl-turns, based on Chou and Fasman probability factors [8]. From the outset it seemed likely that the presence of gMePro at position 3 would stabilise a turn at Pro-'Phe 5. In 3-=MePro-BK, however, a turn is also populated in the region Ser%Arg9. Similarly, in 7-~MePro-
BK, evidence for a turn is seen at Pro:-Phe 5 as well as at Se~-Arg 9. This suggests the possibility that these peptides may fold in a cooperative manner in aqueous solution. This would not be surprising since many of the interactions that stabilise protein structures are known to be cooperative in nature. These and earlier observations [5,6] support the view that substituting proline for at-methylproline may b¢ a general way of stabilising #-turn conformations in linear peptides. Such substitutions are likely to have interesting effects in peptides and proteins where biological activity is intimately linked to conformation. For example, the construction o f peptide libraries on surfaces [20], plastic pegs [21], and beads [22,:231 is currently attracting great interest. Yet many linear peptides of ,~ 5-15 residues based only on the twenty common proteinogenie amino acids may not adopt well 219
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defined secondary structures, a-Methylproline-containing peptides may be useful in such applications, since this analogue can be incorporated using standard solidphase methodologies. There are several cases also where proline residues, and reverse turn conformations, have been implicated in peptide immunogenicity [24-26]. Here again a-methylproline substitutions may lead to peptide analogues with altered, and possibly beneficial immunogenic properties. Based on these considerations, a-methylproline might become more widely useful as a synthetic fl-turn mimetic. Acknowledgement.~: The authors thank the Swiss National Science Foundation (Grant 31-25718.88) and the Kanton of Ztirich for finan. cial support.
REFERENCES [l] Rose. (i.D.. Ciierasch, L.M. and Smith..I.A, 0985) Adv. Prot. Chem. 37, 1-109. [2] Dyson, H.J. and Writ, hi, P.E. (1991) Annu, Rev. Biophys. Biophys. Chem. 20, 519-538. [3] Stewart, D.E,, Sarkar. A. and Wampler..I.E. (1990).I, Mol. Biol. 214. 253-260, [4] MacArthur. M.W. and Thornton, J.M. (1991) .I. Mol, Biol. 218, 397-412, [5] Hinds. M,G., Welsh, J,H., llrennand, D.M., Fisher, L, Glennie, M.J.. Riehards, N.G,.I,, Turner, D.L. and Robinson..I.A. (1991) J. Med. Chem, 34. 1777-1789. [6] Richards, N,G,J., Hinds. M,G,, Brennand. D,M., Glennie, M.J,, Welsh. J.H. and Robinson. J.A. (1990) Bioehem, Pharmacol. 40, 119-123.
[7] Denys, L.. Bothner-By. A,A., Fisher, G,H. and Ryan, J,W, 0982) Biochemistry 21. 6531.
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[8] Chou, P.Y. and Fasman, G.D, (1979) Biophys..I. 26, 367-383. [9] Marion, D. and WCtthrich. K. 0983) Biochem. Biophys. Res. Commun. l l3.967-974. [I0] Braunsehweiler. L, and Ernst, R.R. (1983) J, Mug. Res. 53, 521528. [I l] Bax. A. and Davis, D.G. (1985) J, Mug. Res, 65, 355-360, [12] Bothner-By. A.A,, Stephens, R.L,, Lee, J., Warren. C.D. and Jcanholz, R.W, 0984)J, Am. Chem. Soc. 106, 811-813. [13] WtRhrich, K. (1986) in: NMR of Proteins and Nucleic Acids, J, Wiley. New York. [14] Rance. M., Sorensen, O.W., Bodenhausen, G.. Wal~ner. G., Ernst, R.R.. Wtithrich, K, 0983) Bioehem. Biophys. Res. Commun. 117. 479-485. [15] Lintner, K., Fermandjian, S., St, Pierre, S, and Regoli, D. 0979) Bioehem. Biophys. Res. Commun, 91,803-81 l, [16] Pachler. K.G.R. (1964) Spectrochim. Acta, 20. 581-587. [17] Feeney, .I. (1976) J. MaSh, Res, 21,473-478. [18] Hruby. V.J., in: Chemistry and Biochemistry of Amino Acids, Peptides and Proteins (B. Weinstein, Ed.) vol. 3 Marcel Dekker, New York, 1974, pp. 1-188. [19] London, R.E., Stewart, J.M., Cann, J.R, and Matwiyoff, N.A, 0978) Biochemistry 17, 2270-2277. [20] Fodor, S.P,A., Read, J,L,, Pirrung, M,C,, Stryer, L,, Lu, A.T. and Solas. D. (1991) Science 251. 767-773, [21] Meloen. R.H,, Amerontgen, A.V.. Noort, H.-V., Langedljk, .I,P.M.. Posthumus, W.P,A., Puyk. W.C,. Plasman, H,. Lentra, J.A, and Langeveld, J.P.M. (1991) Annul. Biol. Clin. 49, 231-242. [22] Lain, K.S.. Salmon. S.E., Hcrsh. E.M,. Hruby, V.R., Kazmierski, W.M., Knapp, R,J, (1991) Nature 354, 82-84, [23] Houghten, R.A., Pinilla, C.. Blondelle. S.E., Appel, J.R., Dooley, C.T., Cuervo, J.H. (1991) Nature, 354. 84-86. [24] Javaherian. K.. Langlois, A.J.. LaRosa, G,J,. Profy, A.T,, Bolosnesi. D.P.. Herlihy. W.C,. Putney. S.D. and Matthews. T..l. (1990) Science 250. 1590-1593. [251 Chandrasekhar, K., Profy. A.T. and Dyson. H.J. (1991) Biochemistry 30. 9187-9194. [26] Dyson, H.J,, Lerner. R,A. and Wrisht, P.E. (1988) Annu. Rev. Biophys. Biophys. Chem. 17. 305-324.
