'H- and Natural Abundance "N-NMR Studies of a Derivative of a Rabies Clycoprotein Fragment * HENRIETTE MOLINARI,',' ROBERTO CONSONNI,* MONICA PECNA,3 LUCIA ZETTA,' PAOLO NER1,4 NERI NICCOLA1,4 ALESSANDRA BONC1,4 LUISA LOZZ1,4 MAURO RUSTIC1,4 and MARIA SCARSELLI4 'Dipartimento d i Chimica Organica e Industriale, Via Golgi 19, 20100, 'CNR lstituto di Chimica delle Macromolecole, Laboratorio NMR, Via Ampere 56, 201 31, 31TALFARMAC0, % CNR, lstituto di Chimica delle Macromolecole, Laboratorio NMR, Via Ampere 56, 20131 Milano, and 4CIRE, Centro Didattico Le Scotte, Universit.5 di Siena, 53100 Siena, Italy
SYNOPSIS
Using a combination of one- and two-dimensional methods, 'H- and "N-nmr spectroscopy has been employed to perform the complete assignment and the structural determination of the immunogenic undecapeptide CTTTNSRGTTT in DMSO solution. Nuclear Overhauser enhancement spectroscopy experiments indicated the presence of secondary structures, mainly turn-like structures, which only represent a family, albeit a dominant one, of an ensemble of conformations available to the peptide. Since reverse turns may play an important role as intermediates in protein folding, the experimental observations described here may link the immunological and theoretical approaches to protein folding.
I NTROD U C T 1 0N It has been shown that the binding to muscle nicotinic acetylcholine receptor a t the postsynaptic membrane is an important event of the rabies virus neurotropism.' A large sequence homology between the 190-203 segment of the rabies virus glycoprotein (RVG ) and the putative binding site to the acetylcholine receptor ( AChR) of curare-mimetic snake neurotoxins has been observed.2'3Thus a functional convergence of these distantly related proteins, due to the presence of identical conformational sites, has been suggested. This hypothesis was also supported by the observation that monoclonal antibodies, raised against the 190-203 fragment of RVG, inhibited the binding of RVG and a-bungarotoxin to the nicotinic AChR.4 T h a t the latter antibodies could not recognize the linear peptide was ascribed to the presence, in solution, of a conformational equilibrium where the bioactive reverse turn centered a t the NSRG fragment is scarcely present. The potenBiopolyrners. Vol. :31, 713-723 (1991) (t;1991 John Wiley & Sons, Inc.
CCC 0006-3525/91/060713-11$04.00
* Dedicated to the memory of Antonio De Marco.
' To whom correspondence should be addressed.
tial use of a peptide for the rational design of a synthetic vaccine against the rabies virus involves the stabilization of the structural aspects required for the biological activity. This has been attempted with a multiple solid phase synthesis of 86 analogues, which included the NSRG sequence. The immunoenzymatic essay on the resin-coupled peptides indicated the CTTTNSRGTTT as the smallest analogue that could efficiently bind to the antirabies antib~dies.~ We present evidence here for the presence of secondary structures in this linear, highly immunogenic, free undecapeptide in DMSO solution, determined via one- and two-dimensional 'H- and I5Nnmr techniques.
MATERIALS A N D METHODS Peptide Synthesis T h e CTTTNSRGTTT peptide was synthesized by standard methods and purified by high-performance liquid chromatography on a Vydac CI8 column. 713
714
MOLINARI ET AL.
'H- and "N-NMR Spectroscopy
nomial baseline-correction routine implemented on the X-32 workstation. Phase-sensitive heteronuclear 'H- "N correlations in the inverse mode were performed by employing the heteronuclear multiple-quantum coherence ( HMQC ) sequence modified to record NOE effects as described by Opella et al.'" Two different mixing times of 10 and 180 ms were employed. All 15N experiments were performed on natural abundance 40 m M solutions of the peptide. Spectral width was 5000 Hz in F2 and 2000 Hz in F1,and 94 tl increments with 2560 transients each were acquired to detect NOE effects. For all experiments the delay was 5.25 ms (see Scheme 1 ) .T h e heteronuclear decoupling was achieved employing the Garp-1l 4 decoupling scheme, using a low-power '"N pulse of 94 p s . "N chemical shifts (measured a t 50.68 MHz) were referenced to the 15NH: resonance from 5 M "NH4N03 in 2N H N 0 3 .
Samples were routinely prepared as approximately 9 m M solutions in ( C2H3)2S0.Samples for 15Nexperiments were 40 m M in DMSO. The peptide was also dissolved in water to check the pH, which was 3.09. Spectra were recorded by using a Bruker AM-500 spectrometer. Temperature calibration was performed using methanol chemical shifts and the temperature control unit. All two-dimensional spectra were recorded in the phase-sensitive mode using time-proportional phase in~rementation.~.' Phase-sensitive Hartmann-Hahng ( HOHAHA) experiments, used for spin-system assignment, were performed by using the MLEV-17 composite pulse cycle for the generation of the spin-lock field of rB, = 20 KHz, which was applied during 53 ms. A t the beginning and a t the end of the mixing time two "trim" pulses of 2.5 ms each were applied. Phasesensitive two-dimensional ( 2D ) nuclear Overhauser effect (NOESY) lo experiments were recorded with mixing times of 120 and 200 ms. Spectra were generally recorded with 2048 complex points and 64 or more scans for each free induction decay. Spectral widths were 9-10 ppm in both dimensions and 512 tl values were usually recorded for each 2D spectrum. Spectra were Fourier transformed in the Fz dimension using a phase-shifted sine-bell window function; the final matrix contained 2048 real points in both dimensions. Preliminary experiments were carried out using cryoscopic mixtures (80 : 20 ratio, DMSO : HzO) l 1 in order to solve the amide degeneracy. T h e quality of the HOHAHA and the NOESY spectra was greatly enhanced by the use of a poly-
*'
RESULTS AND DISCUSSION Specific 'H Resonance Assignment
In C T T T N S R G T T T only 6 amino acid types are represented, and there are no aromatic rings. The spectra were recorded in DMSO solutions a t different concentrations and temperatures in the range of 295-310 K. At each temperature the same groups of two or three degenerate protons were identified a t 8.21, 7.86, 4.35, 4.43, 4.1, and 1.5 ppm (see Table I ) . T h e complete identification of the 'H spin systems was achieved for all the amino acid
Table I Sequence-Specific Assignment of CTTTNSRGTTT in DMSO Solution (9 mM) at 295 K" Residue
NH
aCH
PCH
cys-1 Thr-2 Thr-3 Thr-4 Asn-5 Ser-6 Arg-7
8.42 8.79 8.06 7.82 8.21 8.00 8.21
4.35 4.54 4.43 4.35 4.70 4.35 4.35
3.37; 3.07 4.10 4.10 4.10 2.69; 2.53 3.70; 3.64 1.84; 1.59
Gly-8 Thr-9 Thr-10 Thr-11 a
8.15 7.92 7.86 7.86
3.87 4.43 4.27 4.43
7CH andothers
4.10 4.21 4.10
Chemical shifts are in ppm from TMS.
rCH, yCH, yCH,
=
rCH,
=
= =
1.21 1.22 1.22
6CH2
1.59 3.16
rCH, rCH, yCH,
1.22 1.22 = 1.22 = =
Others
SH = 5.11 OH = 5.17 OH = 5.11 OH = 5.11 TNH, = 7.55; 7.05 OH = 5.11 tNH = 7.57 NH2 = 7.30; 7.20; 7.10 OH OH OH
= = =
5.11 5.11 5.11
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
residues ( a t 295 K ) , using HOHAHA’ with different isotropic mixing times, while NOESY experiments gave the sequential assignments. The aliphatic region and the NH-CH, region of the HOHAHA spectrum, are reported in Figure l a and b. Due to the length of the mixing time employed, in all cases, correlations are extended to the entire spin system. The 6 threonines are immediately identified following the correlations of the 6 overlapped methyl groups (occurring in the 1D a t 1.1-1.2 ppm) with the CHP, the CHa, and the corresponding amide resonances, as illustrated in Figure 1b. It is evident that the resonance occurring a t 7.86 ppm corresponds to two threonine amide resonances, showing two cross peaks with a protons a t 4.27 and 4.43 ppm, and two cross peaks with P protons a t 4.21 and 4.10 ppm. The chemical shifts ( 6 ) of threonines together with all other residues are reported in Table I. T h e triplet occurring a t 8.15 ppm (observed in the 1D spectrum) is due to the N H of Gly-8, whose (Y protons occur a t 3.87 ppm. The broad signal a t 8.42 ppm is assigned to the terminal NH2 of the Cys 1, whose a , P’ and P”protons occur a t 4.35,3.37, and 3.07 ppm, respectively. Asn-5 and Ser-6 have the same spin system: P protons of Ser-6 occur a t 3.70 and 3.64 ppm, while protons of Asn-5 occur at higher fields: 2.69 and 2.53 ppm, in agreement with the data reported in the literature (3.88 ppm ~j,,Ser, 2.83 and 2.75 ppm OH Asn) .15 The two coupled broad singlets a t 7.55 and 7.05 ppm are due to Asn 5 N H 2 . Arg-7 is identified because it is the only residue exhibiting a n extended spin system: its N H occurs a t 8.21 ppm and correlates with the a proton at 4 .15 ppm, p‘ and p“ a t 1.84 and 1.59 ppm ( n o stereospecific assignment is given), y’ and y” a t 1.59 ppm, and 6’ and 6” a t 3.16 ppm. The p, y, and 6 protons of Arg-7 further correlate with tNH, a triplet occurring a t 7.57 ppm. An interesting system appears between 7.1 and 7.3 ppm: two very broad resonances nearly completely hidden in the baseline and overlapping with three sharp resonances a t 7.1, 7.2, and 7.3 ppm. We ascribe it to the guanidinium group ot arginine, which can be represented by the following mesomeric forms: NHR IF +
H-N-C=YH Iq I H
H
NER H-N=C-N-H + I
I7
H
NHR
F
Iq
H
around the C-N bond; it has a partial 7r character, as usually observed for amides. The charged NH2 gives rise t o a triplet, owing t o the coupling with 14N (having spin quantum number I = 1) . Indeed, this latter coupling (52 H z ) is detectable only in the presence of a positive charge that lowers the field gradient a t the nitrogen.16 Actually, the analysis of the temperature coefficients suggests the presence of a hydrogen bond a t the N,H2 site. T h e temperature dependence of N H chemical shifts is often used to distinguish between solventshielded and solvent-exposed amide pr0t0ns.l~High As/ A T values are indicative of solvent-exposed N H protons, while low values indicate the involvement of N H in a n intramolecular hydrogen-bonding network. The temperature coefficients obtained in our case, reported in Table 11, are indeed consistent with the involvement of the N,H2 of the guanidinium group in a hydrogen bond. Indeed, the NH2’s have very similar temperature dependencies with A 6 / A T = 1 ppb/K for the triplet centered a t 7.2 ppm and A6/ AT N 1.5 p p b / K for the two broad resonances at 7.3 and 6.95 ppm ( i n this case, the measurement of the temperature coefficient is not very accurate, due to the line broadening). We hypothesize that a hydrogen bond can exist between the Arg-7 NH2 and the carbonyl of Asn-5, so as to mimick the acethylcholine molecule, essential feature for the biological activity observed for this peptide.
Assignments of NOESY Cross Peaks and Evidence for the Presence of Secondary Structures The sequential assignments rely essentially on d n N connectivities obtained through the NOESY experiments, performed with 120- and 200-ms mixing times. These sequential connectivities arise essentially from extended chain conformations of the peptide, while the presence of sequential d“ connectivities, a t the used mixing times (see below), is indicative of folded structures, as pointed out by Dyson.lR The NH-CHa-CHP region, the amide proton region, and the aliphatic region of the NOESY spectrum of the peptide in DMSO are reported in Figure 2a-c. In Figure 2a a n horizontal arrow leads from the N H - a H cross peak of residue i t o the NOESY cross peak indicating the d a N connectivity to residue i 1, and a vertical arrow leads from this NOESY cross peak to the N H - a H cross peak of residue i 1. A good starting point was indeed the Cys-1, since its a proton was unambiguously singled out. The
+
The neutral N,H2 gives rise to two broad resonances due t o the hindered rotation of the two hydrogens
715
+
716
MOLINARI ET AL.
1.5
0
0 0
'
P
0
4.0
O
4-
4.5
5.0 PPm
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Figure 1. HOHAHA spectrum of CTTTNSRGTTT 9 m M in ( C2H3)2S0.Sweep width in F2 was 5000 Hz; 484 tl increments were implemented over 2048 points in t2 dimension. Data matrix was zero filled to 1024 in Fl and a a / 2 shifted sine-bell window was applied in both dimensions; ( a ) CH,-CH, region and ( b ) NH-CH,/CHo region.
sequential assignment went on without difficulty until the end, allowing the complete assignments of the threonines (see Table I ) . Thus the overlapping resonances, in the amide region, are those of Asn-5 and Arg-7 at 8.21 ppm, and of Thr-10 and Thr-11 at 7.86 ppm. A second pathway is due to the NHPH sequential connectivities, which again started from Cys-1 and is represented by arrows in Figure 2a.
Other intraresidue connectivities are observed among the Arg-7 tNH and Arg-7 6, y,/? CH2,among Asn-5 NH2 and its ,6 protons, and between the NH of threonines and their methyl groups. One longer range interresidue NOE is observed between the CH3 of Thr-4 and the NH of Asn-5. In the amide proton region of the NOESY spectrum (Figure 2b) NOE cross peaks are observed among the amide proton resonances of all the sequentially adjacent amino
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
T2
R7 S6 N5 T3 1
Cl
Y
Y
N! N[
717
experiment was performed with a shorter (120-ms) mixing time and it exhibited the same cross-peak pattern. T h e correlation time, calculated as described by Esposito e t al.," exploiting the known distance between a pair of protons was indeed estimated to be 1.3 ns. In these conditions the spin diffusion effects are negligible.20~21 The occurrence of NOES between the amide proton resonances of several sequential amino acid residues, together with the low values of the temperature coefficients measured for Cys-1 NH2, Thr-4 NH, and Arg-7 t N H and vNH2, suggests the presence of a population of folded structures in the conformational ensemble. Longer range interresidue NOE connectivities are observed between the cuH proton of Cys-1 and the N H resonance of Thr-3 (this cross peak is very weak, and visible when very low contour-plot levels of 2D NOESY are plotted), and between the a H resonance of Thr-2 and the N H resonance of Thr-4 (Figure 2 a ) . Even if present, no other d o N ( i ,i 2 ) NOE connectivities could have been observed because of resonance overlapping. The observation of few d,N(i, i 2 ) NOE connectivities supports the existence of local structures with short-range order. Our observations are in agreement with previous nmr studies, which establish that sequential d" NOE connectivities are seen only in those regions of peptides that preferentially adopt folded conformations. Finally, we note that the secondary structure does not appear as arising through aggregation, since the chemical shifts of the amide resonances are independent of peptide concentration between 9 m M and 200 p M .
+
+
GI
B a -4.5
ba
N!
,
PPm
,
,
, 8.5
-5.0
SH ,
,
,
,
, 8.0
I
,
,
,
,
tppm
7.5
Figure 1. (continued from the previous page)
Table I1 NMR Parameters for Proton Amides of CTTTNSRGTTT in DMSO Solution Amino Acid
acid residues, until Thr-9, as indicated by the arrows. T h e connectivity between this residue and the following overlapping threonines 10 and 11 cannot be precisely detected because the cross peak is too close to the diagonal. It must be noticed that, following this pathway, one cross peak has double intensity and represents the connectivity between Asn-5 / Ser6 and Ser-6/Arg-7, due to the overlapping Asn-5 and Arg-7 amide protons. In view of the strong NOE cross peak observed between the u H protons and N H protons, it was important to establish that the NH-NH cross peaks did not arise from spin diffu~ion.'~ Another NOESY
JCHNH
cys-1 Thr-2 Thr-3 Thr-4 Asn-5
7.02 8.24 8.24 7.30
Ser-6 Arg-7
7.15 7.30
Gly-8 Thr-9 Thr-10 Thr-11
8.24 8.24(Thr-11) 7.80(Thr-10)
A6/AT 1.0 5.6 4.3 3.0 4.O(Arg-7)
4.0 5.3 4.0 4.6(Asn-5) 2.3 1.0 3.3 5.0 4.6( T h r - 11) 4.0(Thr-lO)
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MOLINARI ET AL.
NH I
I
I
I1
11
-3.5
-4.0
-4.5
O 5 -5.0 I
PPm
1
1
I
I
8.5
I
I
I
I
,
/
,
I
PPm
7.5
8.0
T2
3CT
rpn
c ?
8
'0
3
Figure 2. Phase-sensitive NOESY spectrum of CTTTNSRGTTT 9 m M in ( C2H,)sS0. Mixing time was 120 ms; sweep width in F2was 6410 Hz; 512 tl increments were implemented over 2048 points in t2dimension. Data matrix was zero filled to 1024 in Fl and a T / 2 shifted sine-bell window was applied in both dimensions; ( a ) NH-CHJCH, region, the asterisk indicates the daN(i, i 2 ) connectivity between the Thr-2 and Thr-4, ( b ) NH-NH region, and ( c ) CH,-CH, region.
+
A set of distances was obtained (see Table 111) from NOESY data employing the initial rate approximation method, l5 by using the fixed distance of the methylene protons in Asn-5 and Arg-7 as internal calibration. T h e observation of strong d a N ( i, i 1) NOE connectivities over the entire peptide means that the
+
conformational ensemble of the peptide includes a population of extended chain forms, which appear as predominating in the unfolded structures." In addition, d N Ni, ( i 1) and few dnN(i, i 2 ) NOE connectivities are observed they indicate the presence of a pronounced preference for folded conformations. T h e consecutive d"(i, i 1) NOE con-
+
+
+
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
719
:.5
2.0
0
2.5 4
0 3.0
0 8
3
3.5
0 4.0
4.5
Q
I
bJ
@-
0
5.0 PPm PPm
4.5
4.0
3.5
Figure 2.
+
2.5
2.0
(continued from the previous page)
nectivities are as would be expected for a n a- or 310helix2'; however, none of the characteristic d n N ( i , i 3 ) , or d,,$(i, i 3 ) NOE connectivities, diagnostic of a regular helix, were observed. On the other hand, the observed high values of the NH-aH coupling constants, JNH.aH, for well-resolved NH protons in the 1D spectrum (see Table 11),are not in agreement with the expected low values 3 J ~ ~ = 4. Hz, c ychar~ acteristic of a - or 310-helices.'5Nor are the data consistent with the presence of P-sheet elements, where d" connectivities are not usually detected, and 3 J N ~ - a=~ 9 Hz, which is not the present case.
+
3.0
+
2 ) connectivities indicate the T h e d,N(i, i presence of local structures and short-range order. We also notice the presence of d o N ( i, i 1) connectivities, which are commonly observed in 0-turns and in helical structures.22 Turns of two general classes, type I and type 11, are possible (type I11 turns are very similar to type I)23;these two types could not be precisely distinguished by the present experiments. Type I and type I1 turns can, in principle, be distinguished on the basis of NOE connectivities and the 3 J ~ coupling , constants a t positions 2 and 3 of the Unfortunately for flexible
+
720
MOLINARI ET AL.
Table I11 Proton Distances Obtained from NMR Data cys-1
Ha-NH(Thr-2) Ha-NH(Thr-3)
2.24 3.39
HP1-NH(Thr-2)
3.29
3.12
Thr-2 NH-NH(Thr-3) Ha-NH
3.11 2.64
Ha-NH(Thr-3) HP-NH
2.10 2.84
Ha-NH(Thr-4) Hr-NH
3.45 2.70
2.16 2.58
NH-Hn
2.63
3.06 2.72
HP2-NH(Ser-6)
3.08
3.22 2.83
NH-Ha
2.47
Thr-3 NH-NH(Thr-4) NH-HB
2.69 2.61
Ha-NH(Thr-4) NH-Hy Thr-4
HP-NH(Asn-5)
2.71
NH-Ha
2.71 Asn-5
Ha-NH(Ser-6) NH-HB1
2.20 2.83
HP1-NH(Ser-6) NH-HP2 Ser-6
HP1-NH(Arg-7) NH-HP1
3.15 2.83
HP2-NH(Arg-7) NH-HPB Arg-7
Ha-NH(Gly-8)
2.20
NH-H-/l
3.04 Gly-8
NH-NH(Thr-9)
2.82
Ha-NH(Thr-9)
linear peptides in solution, the coupling constants represent a n ensemble average and are of less value than other parameters in distinguishing between classes of 0-turns. A large population is necessary in order that a significantly lower coupling constant is observed, since the value for fully folded turns is 5 H z . Our ~ ~ nmr data slightly favor a type I1 turn, in that strong d o N ( i, i f 1)NOE connectivities are observed, characteristic of type I1 t ~ r n . ~ ~ , ’ ~ Thus nmr data all together seem t o indicate that the @-turnsare one important family of a n ensemble of conformations available to the peptide. T h e peptide then adopts a number of transient local conformations, the turn-like structures, all of which may interconvert rapidly by way of a n extended “random coil” form. In agreement with the results recently reported by Dyson et al., 27 this ensemble of secondary structures could be defined as a “nascent helix.” As reported by D y ~ o n , ’the ~ NOEs for a flexible peptide are a population-weighted average of the
2.32
NOEs in all of the conformational states available t o the peptide. For short linear peptides in solution, the d” ( i , i 1)distance becomes just short enough to give observable NOEs between residues with @ and \k angles in the helix region of (@, \k) space. Further, dNNdoes not vary much over the allowed range of @ and \k angles in this region. Thus differences in intensity of the NH-NH NOEs reflect differences in the population of “helical conformers” a t each residue, once the decreased correlation times a t the ends of the peptide are taken into account. In CTTTNSRGTTT, NOEs of similar intensities are observed throughout the sequence, the most intense peak being observed for the dipolar correlation between T h r - 3 and Thr-4, suggesting that the highest population of structured conformers occurs a t these residues (Thr-4 also exhibits a low-temperature coefficient). Indeed, a preliminary conformational analysis, performed using the SEARCH program of the SYBYL software, indicated a lack of
+
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
convergence t o a unique family of structures, in agreement with the presence of the proposed conformational equilibrium. Experiments in cryoscopic mixtures (DMSO / water) a t low temperatures are under study, with the aim of obtaining a number of distance constraints appropriate for dynamic simulations. 15NSpectroscopy
In view of the importance of structural studies on short synthetic immunogenic peptides, I5N experiments have also been performed. T h e use of 15N, which has a larger chemical shift dispersion than its proton counterpart, has been shown t o be a very useful method for alleviating the problem of spectral overlap. In addition, correlation between 15N chemical shifts and structural features could be of great interest. T o maximize sensitivity, the I5N spectrum is typically measured indirectly, as the F1 dimension of a 2D experiment in which the 'H magnetization is both initially excited and detected. In the experiments shown here, heteronuclear 'H- 15N multiplequantum coherence (HMQC) '*is allowed to evolve during the evolution period tl . Recently, a new experiment has been described that combines heteronuclear correlation and 'H homonuclear NOESY to give 2D spectra with cross peaks between 'H/15N correlation peaks from amide sites and other protons dipolarly coupled to them. The original experiment was performed on uniformly 15N-labeled samples. We report here a study on the applicability of this experiment to our peptide at natural 15Nabundance. T h e sequence employed is depicted in Scheme 1: 13928
90'
180'
90
Scheme 1
T h e HMQC present during the tl evolution period evolves only due to 15Nchemical shifts with respect to tl and consists both of zero- and double-quantum coherences interconverted by the 180" ('H) pulse. After the last 90" ( 15N),which converts the zeroand double-quantum coherences into antiphase detectable proton magnetization, a delay A = 1 /2 J cL5 N. 1H followed ) by a 90" ('H) pulse brings the proton magnetization along the z axis, allowing
721
a n exchange of magnetization between dipolarly coupled protons, during the mixing time 7,. Heteronuclear decoupling during t 2 is mandatory in these kinds of experiments. It is achieved through composite pulse schemes, such as MLEV-4, 29 WALTZ-16,30or GARP-1.14 Their performance, as far as the frequency range of the decoupling is concerned, has been reported.14 The GARP-1 scheme appears to offer the best performance, but the WALTZ-16 scheme is efficient enough when dealing with 15N,which is usually recorded with small sweep width. When the heteronuclear-NOE experiment was performed with a short mixing interval (i.e., 7 , = 10 m s ) , each amide site gave rise to a single 'H/15N cross peak, as shown in Figure 3a. Since it is impossible to distinguish between the overlapping proton resonances of Thr-10 and Thr-11, the assignment of nitrogen resonances, only based on the simple HMQC experiment (Figure 3 a ) , was not complete. For the other two overlapping resonances of Asn-5 and Arg-7, a unique correlation is observed in the heterocorrelated experiment (Figure 3 a ) , indicating that their nitrogen resonances are also degenerate. The analysis of the experiment performed with a longer mixing time (7, = 180 ms; Fig. 3b) reveals additional peaks due to the dipolar correlation between each N H with its a proton. Since the experiment was performed on a 40 m M peptide solution, a NOESY on the same concentrated solution was additionally performed, to check for the proton assignment. From the experiment of Figure 3b it is possible to distinguish between Thr-10 and Thr-11, which correlate with a H s occurring a t different chemical shifts-an indication of the potentiality of this kind of experiment for 15N assignment. All the other 15N assignments were confirmed. T h e analysis of cross sections revealed the presence of cross peaks in the amide region as well, one between the Thr-3 and Thr-4, thus confirming the previous hypothesis of a highest population of structured conformers occurring a t these residues, and a weak one between Thr-4 and Asn-5. I t is apparent that in this experiment the strong intraresidue NOES are observed, while only a couple of NHN H connectivities could be detected from the analysis of cross sections. This deserves some remarks. T h e HMQC sequence belongs t o the class of pulse schemes in which the satellite signals are selected by phase cycling alone3' and is therefore well suited to detect enriched signals. The stringent requirements of stability and dynamic range imposed on the nmr spectrometer when the signals selection is accomplished by phase cycling only, together with
722
MOLINARI ET AL.
105
1
110
i'
I
3
-
45 77
I
1H
ppn
8.0
6.0
7.0
510
410
1H
Figure 3. Phase-sensitive heteronuclear 'H- 15N spectrum of CTTTNSRGTTT obtained by employing the HMQC sequence modified to record NOE effects. The experiment was performed on a natural 15Nabundance, 40 m M in ( C2H3)2SO;sweep width was 5000 and 2000 Hz in F2 and F, , respectively; 94 tl increments, each of 2560 scans, were implemented over 2048 points in t2 dimension. Data matrix was zero filled to 512 in F , and a ?r/2 shifted sine-bell window was applied in both dimensions; ( a ) mixing time 10 ms, and ( b ) mixing time 180 ms.
the incomplete suppression of the main signals from the protons bound t o 14N, decrease the sensitivity of the HMQC scheme when applied to natural abundance samples. In addition, the HMQC exhibits in F1the homonuclear multiplet of its active protons and the effective linewidth will be unfavorable if compared to single quantum coherence experiments, as recently r e p ~ r t e d . ~All " ~these ~ factors are certainly crucial in a sensitive experiment as the detection of small NOE effects in a reverse pulse scheme, and this could explain the lack of the weaker NH-NH cross peaks from the experiment of Figure 3b. It is worth mentioning that, in the described heteronuclear-NOE experiment applied to natural abundance 15N samples, a much higher number of transients, with respect to those necessary to obtain a normal HMQC correlation, is necessary for each tl . As a result, quite long overall acquisition times are required (3-4 days). In spite of this drawback, it is still very advantageous to get 15N spectra with unprohibitive amounts of biological sample. In order to improve the performance of the described method we are presently studying other sequences, based on
the evolution of 15N single quantum coherence, on which pulses for NOE detection are superimposed.
CONCLUSIONS T h e combined use of 'H and 15N high-field nmr spectroscopy provided the complete assignment of both proton and nitrogen resonances for the undecapeptide CTTTNSRGTTT in DMSO solution. In particular, the heteronuclear-NOE reverse experiment offers the possibility of assigning unambiguously the "N spectra, irrespective of overlapping in the proton resonances. 15N data may play a n important role in view of studies on synthetic immunogenic peptides bound to antibodies. Structural data obtained from NOESY experiments agree with the presence of ordered structures in the peptide. T h e observation that this short immunogenic peptide fragment may adopt ordered structures bears significantly on several fundamental issues, including the problem of protein folding.z3x33Stable local structures, which may be different from those in the final folded protein, can be
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
recognized by antibodies, and have been postulated as initiation sites in f ~ l d i n g . The ~ ~ -experimental ~~ observations described here may thus link the immunological and theoretical approaches to protein folding, and show that the combined use of synthetic immunogenic fragments of proteins and high-field nmr spectroscopy provides a powerful approach to the identification of local structures of biological relevance. We thank Giulio Zannoni and Fulvia Greco for their expert technical assistance.
REFERENCES 1. Lentz, T. L., Burrage, T. G., Smith, A. L., Crick, J. & Tignor, T. H. (1982) Science 215, 182-184. 2. Lentz, T. L., Wilson, P. T., Hawrot, E. & Speicher, 11. W. (1984) Science 226,847-848. 3. Neri, P., Bracci, L., Di Tommaso, A., Lozzi, L., Rustici,
M., Santucci, A., Soldani, P., Petreni, S., Niccolai, N., Mascagni, P., Siligardi, G. & Gibbons, W. A. (1987) in Macromolecular Biorecognition. Principles and Methods, Chaiken I., Chiancone E., Fontana A. & Neri P., Eds., Humana Press, Clifton, NJ, pp. 259-272. 4. Rracci, L., Antoni, G., Cusi, M. G., Lozzi, L., Niccolai, N., Petreni, S., Rustici, M., Santucci, A., Soldani, P., Valensin, P. E. & Neri, P. (1988) Mol. Immunol. 25, 881-888. 5. Lozzi, L., Rustici, M., Bracci, L., Petreni, S., Santucci, A., Soldani, P., Spreafico, A. & Neri, P., to he puh-
lished. 6. Barany, G., Kneb-Cordonier, N. & Mullen, D. J . ( 1987) Int. J . Pept. Protein Res. 30, 705-734. 7. Drobny, G., Pines, A., Sinton, S., Weitekamp, D. & Wemmer, D. ( 1979) Symp. Faraday SOC.13, 49-55. 8. Marion, D. & Wuethrich, K. (1983) Biochem. Biophys. Rex Commun. 113, 967-974. 9. Bax, A. & Davis, D. J. (1985) J . Magn. Reson. 63, 207-212. 10. ,Jeener, J., Meier, B. H., Bachmann, P. & Ernst, R. K.( 1979) J . Chem. Phys. 71,4546-4553. 11. Motta, A., Picone, D., Tancredi, T. & Temussi, P. A. ( 1987) J . Magn. Reson. 75, 364-370. 12. Bax, A., Griffey, R. H. & Hawkins, B. L. (1983) J. Magn. Reson. 55, 301-312. 13. Shon, K. & Opella, S. (1989) J. Magn. Reson. 82, 193- 195. 14. Shaka, A. J., Barker, P. B. & Freeman, R. (1985) J. Magri. Reson. 64, 547-550.
723
15. Wuethrich, K. ( 1986) N M R of Proteins and Nucleic Acids, John Wiley & Sons, New York. 16. Webb, G. A. & Witanowski, M. ( 1973) Nitrogen N M R ,
Plenum Press, London. 17. Kessler, H. (1982) Angew. Chem. Znt. Ed. Engl. 21, 5 12-523. 18. Dyson, H. J., Rance, M., Houghten, R. A., Lerner, R. A. & Right, P. E. (1988) J . Mol. B i d . 2 0 1 , 161200. 19. Kalk, A. & Berendsen, H. J. (1976) J . Magn. Reson. 24, 343-366. 20. Esposito, G. & Pastore, A. (1988) J. Magn. Reson. 76,331-336. 21. Neuhaus, D. & Williamson, M. P. (1989) The Nuclear Overhauser Effect in Structural and Conformational Analysis, VCH Publishers, pp. 89-94. 22. Wuethrich, K., Billeter, M. & Braun, W. (1984) J . Mol. Biol. 180, 715-740. 23. Richardson, J. S. (1981) Adv. Protein Chem. 34,167339. 24. Wagner, G., Neuhaus, D., Worgotter, E., Vasak, M., Kagi, J. H. R. & Wuethrich, K. (1986) J . Mol. Biol. 187, 131-135. 25. Shenderovich, M. D., Nikiforovich, G. V. & Chipens, G. I. (1984) J. Magn. Reson. 59,l-12. 26. Rose, G. D., Gierasch, L. M. & Smith, J . A. (1985) Adu. Protein Chem. 37, 1-105. 27. Dyson, H. J., Rance, M., Houghten, R. A., Wright, P. E. & Lerner, R. A. (1988) J. Mol. B i d . 201, 201217. 28. Nonvood, T. J., Boyd, J., Heritage, J. E.. Soffe, N. & Campbell, I. D. ( 1990) J. Magn. Reson. 87,488-501. 29. Levitt, M. H., Freeman, R. & Frenkiel, T. (1982) J. Magn. Reson. 50, 157-163. 30. Shaka, J. A., Keeler, J. & Freeman, R. ( 1983) J . Magn. Reson. 53, 313-327. 31. Otting, G. & Wuethrich, K. (1988) J . Magn. Reson. 76,569-577. 32. Bax, A., Ikura, M., Kay, L. E., Torchia, D. A. & Tschudin, R. ( 1990) J. Magn. Reson. 8 6 , 304-318. 33. Binkelstein, A. V. & Ptitsyn, 0. B. ( 1986) J . Mol. Biol. 103, 15-24. 34. Teale, J. M. & Benjamin, D. C. (1976) J . Hiol. Chem. 251, 4609-4615. 35. Celada, F., Fowler, A. V. & Zabin, I. ( 1978) Biochemistry 17,5156-5160. 36. Anfinsen, C. B. & Scheraga, H. A. ( 1975) Adu. Protein Chem. 29, 205-300.
Received July 20, 1990 Accepted December 5, 1990
SYNOPSIS
Using a combination of one- and two-dimensional methods, 'H- and "N-nmr spectroscopy has been employed to perform the complete assignment and the structural determination of the immunogenic undecapeptide CTTTNSRGTTT in DMSO solution. Nuclear Overhauser enhancement spectroscopy experiments indicated the presence of secondary structures, mainly turn-like structures, which only represent a family, albeit a dominant one, of an ensemble of conformations available to the peptide. Since reverse turns may play an important role as intermediates in protein folding, the experimental observations described here may link the immunological and theoretical approaches to protein folding.
I NTROD U C T 1 0N It has been shown that the binding to muscle nicotinic acetylcholine receptor a t the postsynaptic membrane is an important event of the rabies virus neurotropism.' A large sequence homology between the 190-203 segment of the rabies virus glycoprotein (RVG ) and the putative binding site to the acetylcholine receptor ( AChR) of curare-mimetic snake neurotoxins has been observed.2'3Thus a functional convergence of these distantly related proteins, due to the presence of identical conformational sites, has been suggested. This hypothesis was also supported by the observation that monoclonal antibodies, raised against the 190-203 fragment of RVG, inhibited the binding of RVG and a-bungarotoxin to the nicotinic AChR.4 T h a t the latter antibodies could not recognize the linear peptide was ascribed to the presence, in solution, of a conformational equilibrium where the bioactive reverse turn centered a t the NSRG fragment is scarcely present. The potenBiopolyrners. Vol. :31, 713-723 (1991) (t;1991 John Wiley & Sons, Inc.
CCC 0006-3525/91/060713-11$04.00
* Dedicated to the memory of Antonio De Marco.
' To whom correspondence should be addressed.
tial use of a peptide for the rational design of a synthetic vaccine against the rabies virus involves the stabilization of the structural aspects required for the biological activity. This has been attempted with a multiple solid phase synthesis of 86 analogues, which included the NSRG sequence. The immunoenzymatic essay on the resin-coupled peptides indicated the CTTTNSRGTTT as the smallest analogue that could efficiently bind to the antirabies antib~dies.~ We present evidence here for the presence of secondary structures in this linear, highly immunogenic, free undecapeptide in DMSO solution, determined via one- and two-dimensional 'H- and I5Nnmr techniques.
MATERIALS A N D METHODS Peptide Synthesis T h e CTTTNSRGTTT peptide was synthesized by standard methods and purified by high-performance liquid chromatography on a Vydac CI8 column. 713
714
MOLINARI ET AL.
'H- and "N-NMR Spectroscopy
nomial baseline-correction routine implemented on the X-32 workstation. Phase-sensitive heteronuclear 'H- "N correlations in the inverse mode were performed by employing the heteronuclear multiple-quantum coherence ( HMQC ) sequence modified to record NOE effects as described by Opella et al.'" Two different mixing times of 10 and 180 ms were employed. All 15N experiments were performed on natural abundance 40 m M solutions of the peptide. Spectral width was 5000 Hz in F2 and 2000 Hz in F1,and 94 tl increments with 2560 transients each were acquired to detect NOE effects. For all experiments the delay was 5.25 ms (see Scheme 1 ) .T h e heteronuclear decoupling was achieved employing the Garp-1l 4 decoupling scheme, using a low-power '"N pulse of 94 p s . "N chemical shifts (measured a t 50.68 MHz) were referenced to the 15NH: resonance from 5 M "NH4N03 in 2N H N 0 3 .
Samples were routinely prepared as approximately 9 m M solutions in ( C2H3)2S0.Samples for 15Nexperiments were 40 m M in DMSO. The peptide was also dissolved in water to check the pH, which was 3.09. Spectra were recorded by using a Bruker AM-500 spectrometer. Temperature calibration was performed using methanol chemical shifts and the temperature control unit. All two-dimensional spectra were recorded in the phase-sensitive mode using time-proportional phase in~rementation.~.' Phase-sensitive Hartmann-Hahng ( HOHAHA) experiments, used for spin-system assignment, were performed by using the MLEV-17 composite pulse cycle for the generation of the spin-lock field of rB, = 20 KHz, which was applied during 53 ms. A t the beginning and a t the end of the mixing time two "trim" pulses of 2.5 ms each were applied. Phasesensitive two-dimensional ( 2D ) nuclear Overhauser effect (NOESY) lo experiments were recorded with mixing times of 120 and 200 ms. Spectra were generally recorded with 2048 complex points and 64 or more scans for each free induction decay. Spectral widths were 9-10 ppm in both dimensions and 512 tl values were usually recorded for each 2D spectrum. Spectra were Fourier transformed in the Fz dimension using a phase-shifted sine-bell window function; the final matrix contained 2048 real points in both dimensions. Preliminary experiments were carried out using cryoscopic mixtures (80 : 20 ratio, DMSO : HzO) l 1 in order to solve the amide degeneracy. T h e quality of the HOHAHA and the NOESY spectra was greatly enhanced by the use of a poly-
*'
RESULTS AND DISCUSSION Specific 'H Resonance Assignment
In C T T T N S R G T T T only 6 amino acid types are represented, and there are no aromatic rings. The spectra were recorded in DMSO solutions a t different concentrations and temperatures in the range of 295-310 K. At each temperature the same groups of two or three degenerate protons were identified a t 8.21, 7.86, 4.35, 4.43, 4.1, and 1.5 ppm (see Table I ) . T h e complete identification of the 'H spin systems was achieved for all the amino acid
Table I Sequence-Specific Assignment of CTTTNSRGTTT in DMSO Solution (9 mM) at 295 K" Residue
NH
aCH
PCH
cys-1 Thr-2 Thr-3 Thr-4 Asn-5 Ser-6 Arg-7
8.42 8.79 8.06 7.82 8.21 8.00 8.21
4.35 4.54 4.43 4.35 4.70 4.35 4.35
3.37; 3.07 4.10 4.10 4.10 2.69; 2.53 3.70; 3.64 1.84; 1.59
Gly-8 Thr-9 Thr-10 Thr-11 a
8.15 7.92 7.86 7.86
3.87 4.43 4.27 4.43
7CH andothers
4.10 4.21 4.10
Chemical shifts are in ppm from TMS.
rCH, yCH, yCH,
=
rCH,
=
= =
1.21 1.22 1.22
6CH2
1.59 3.16
rCH, rCH, yCH,
1.22 1.22 = 1.22 = =
Others
SH = 5.11 OH = 5.17 OH = 5.11 OH = 5.11 TNH, = 7.55; 7.05 OH = 5.11 tNH = 7.57 NH2 = 7.30; 7.20; 7.10 OH OH OH
= = =
5.11 5.11 5.11
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
residues ( a t 295 K ) , using HOHAHA’ with different isotropic mixing times, while NOESY experiments gave the sequential assignments. The aliphatic region and the NH-CH, region of the HOHAHA spectrum, are reported in Figure l a and b. Due to the length of the mixing time employed, in all cases, correlations are extended to the entire spin system. The 6 threonines are immediately identified following the correlations of the 6 overlapped methyl groups (occurring in the 1D a t 1.1-1.2 ppm) with the CHP, the CHa, and the corresponding amide resonances, as illustrated in Figure 1b. It is evident that the resonance occurring a t 7.86 ppm corresponds to two threonine amide resonances, showing two cross peaks with a protons a t 4.27 and 4.43 ppm, and two cross peaks with P protons a t 4.21 and 4.10 ppm. The chemical shifts ( 6 ) of threonines together with all other residues are reported in Table I. T h e triplet occurring a t 8.15 ppm (observed in the 1D spectrum) is due to the N H of Gly-8, whose (Y protons occur a t 3.87 ppm. The broad signal a t 8.42 ppm is assigned to the terminal NH2 of the Cys 1, whose a , P’ and P”protons occur a t 4.35,3.37, and 3.07 ppm, respectively. Asn-5 and Ser-6 have the same spin system: P protons of Ser-6 occur a t 3.70 and 3.64 ppm, while protons of Asn-5 occur at higher fields: 2.69 and 2.53 ppm, in agreement with the data reported in the literature (3.88 ppm ~j,,Ser, 2.83 and 2.75 ppm OH Asn) .15 The two coupled broad singlets a t 7.55 and 7.05 ppm are due to Asn 5 N H 2 . Arg-7 is identified because it is the only residue exhibiting a n extended spin system: its N H occurs a t 8.21 ppm and correlates with the a proton at 4 .15 ppm, p‘ and p“ a t 1.84 and 1.59 ppm ( n o stereospecific assignment is given), y’ and y” a t 1.59 ppm, and 6’ and 6” a t 3.16 ppm. The p, y, and 6 protons of Arg-7 further correlate with tNH, a triplet occurring a t 7.57 ppm. An interesting system appears between 7.1 and 7.3 ppm: two very broad resonances nearly completely hidden in the baseline and overlapping with three sharp resonances a t 7.1, 7.2, and 7.3 ppm. We ascribe it to the guanidinium group ot arginine, which can be represented by the following mesomeric forms: NHR IF +
H-N-C=YH Iq I H
H
NER H-N=C-N-H + I
I7
H
NHR
F
Iq
H
around the C-N bond; it has a partial 7r character, as usually observed for amides. The charged NH2 gives rise t o a triplet, owing t o the coupling with 14N (having spin quantum number I = 1) . Indeed, this latter coupling (52 H z ) is detectable only in the presence of a positive charge that lowers the field gradient a t the nitrogen.16 Actually, the analysis of the temperature coefficients suggests the presence of a hydrogen bond a t the N,H2 site. T h e temperature dependence of N H chemical shifts is often used to distinguish between solventshielded and solvent-exposed amide pr0t0ns.l~High As/ A T values are indicative of solvent-exposed N H protons, while low values indicate the involvement of N H in a n intramolecular hydrogen-bonding network. The temperature coefficients obtained in our case, reported in Table 11, are indeed consistent with the involvement of the N,H2 of the guanidinium group in a hydrogen bond. Indeed, the NH2’s have very similar temperature dependencies with A 6 / A T = 1 ppb/K for the triplet centered a t 7.2 ppm and A6/ AT N 1.5 p p b / K for the two broad resonances at 7.3 and 6.95 ppm ( i n this case, the measurement of the temperature coefficient is not very accurate, due to the line broadening). We hypothesize that a hydrogen bond can exist between the Arg-7 NH2 and the carbonyl of Asn-5, so as to mimick the acethylcholine molecule, essential feature for the biological activity observed for this peptide.
Assignments of NOESY Cross Peaks and Evidence for the Presence of Secondary Structures The sequential assignments rely essentially on d n N connectivities obtained through the NOESY experiments, performed with 120- and 200-ms mixing times. These sequential connectivities arise essentially from extended chain conformations of the peptide, while the presence of sequential d“ connectivities, a t the used mixing times (see below), is indicative of folded structures, as pointed out by Dyson.lR The NH-CHa-CHP region, the amide proton region, and the aliphatic region of the NOESY spectrum of the peptide in DMSO are reported in Figure 2a-c. In Figure 2a a n horizontal arrow leads from the N H - a H cross peak of residue i t o the NOESY cross peak indicating the d a N connectivity to residue i 1, and a vertical arrow leads from this NOESY cross peak to the N H - a H cross peak of residue i 1. A good starting point was indeed the Cys-1, since its a proton was unambiguously singled out. The
+
The neutral N,H2 gives rise to two broad resonances due t o the hindered rotation of the two hydrogens
715
+
716
MOLINARI ET AL.
1.5
0
0 0
'
P
0
4.0
O
4-
4.5
5.0 PPm
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Figure 1. HOHAHA spectrum of CTTTNSRGTTT 9 m M in ( C2H3)2S0.Sweep width in F2 was 5000 Hz; 484 tl increments were implemented over 2048 points in t2 dimension. Data matrix was zero filled to 1024 in Fl and a a / 2 shifted sine-bell window was applied in both dimensions; ( a ) CH,-CH, region and ( b ) NH-CH,/CHo region.
sequential assignment went on without difficulty until the end, allowing the complete assignments of the threonines (see Table I ) . Thus the overlapping resonances, in the amide region, are those of Asn-5 and Arg-7 at 8.21 ppm, and of Thr-10 and Thr-11 at 7.86 ppm. A second pathway is due to the NHPH sequential connectivities, which again started from Cys-1 and is represented by arrows in Figure 2a.
Other intraresidue connectivities are observed among the Arg-7 tNH and Arg-7 6, y,/? CH2,among Asn-5 NH2 and its ,6 protons, and between the NH of threonines and their methyl groups. One longer range interresidue NOE is observed between the CH3 of Thr-4 and the NH of Asn-5. In the amide proton region of the NOESY spectrum (Figure 2b) NOE cross peaks are observed among the amide proton resonances of all the sequentially adjacent amino
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
T2
R7 S6 N5 T3 1
Cl
Y
Y
N! N[
717
experiment was performed with a shorter (120-ms) mixing time and it exhibited the same cross-peak pattern. T h e correlation time, calculated as described by Esposito e t al.," exploiting the known distance between a pair of protons was indeed estimated to be 1.3 ns. In these conditions the spin diffusion effects are negligible.20~21 The occurrence of NOES between the amide proton resonances of several sequential amino acid residues, together with the low values of the temperature coefficients measured for Cys-1 NH2, Thr-4 NH, and Arg-7 t N H and vNH2, suggests the presence of a population of folded structures in the conformational ensemble. Longer range interresidue NOE connectivities are observed between the cuH proton of Cys-1 and the N H resonance of Thr-3 (this cross peak is very weak, and visible when very low contour-plot levels of 2D NOESY are plotted), and between the a H resonance of Thr-2 and the N H resonance of Thr-4 (Figure 2 a ) . Even if present, no other d o N ( i ,i 2 ) NOE connectivities could have been observed because of resonance overlapping. The observation of few d,N(i, i 2 ) NOE connectivities supports the existence of local structures with short-range order. Our observations are in agreement with previous nmr studies, which establish that sequential d" NOE connectivities are seen only in those regions of peptides that preferentially adopt folded conformations. Finally, we note that the secondary structure does not appear as arising through aggregation, since the chemical shifts of the amide resonances are independent of peptide concentration between 9 m M and 200 p M .
+
+
GI
B a -4.5
ba
N!
,
PPm
,
,
, 8.5
-5.0
SH ,
,
,
,
, 8.0
I
,
,
,
,
tppm
7.5
Figure 1. (continued from the previous page)
Table I1 NMR Parameters for Proton Amides of CTTTNSRGTTT in DMSO Solution Amino Acid
acid residues, until Thr-9, as indicated by the arrows. T h e connectivity between this residue and the following overlapping threonines 10 and 11 cannot be precisely detected because the cross peak is too close to the diagonal. It must be noticed that, following this pathway, one cross peak has double intensity and represents the connectivity between Asn-5 / Ser6 and Ser-6/Arg-7, due to the overlapping Asn-5 and Arg-7 amide protons. In view of the strong NOE cross peak observed between the u H protons and N H protons, it was important to establish that the NH-NH cross peaks did not arise from spin diffu~ion.'~ Another NOESY
JCHNH
cys-1 Thr-2 Thr-3 Thr-4 Asn-5
7.02 8.24 8.24 7.30
Ser-6 Arg-7
7.15 7.30
Gly-8 Thr-9 Thr-10 Thr-11
8.24 8.24(Thr-11) 7.80(Thr-10)
A6/AT 1.0 5.6 4.3 3.0 4.O(Arg-7)
4.0 5.3 4.0 4.6(Asn-5) 2.3 1.0 3.3 5.0 4.6( T h r - 11) 4.0(Thr-lO)
718
MOLINARI ET AL.
NH I
I
I
I1
11
-3.5
-4.0
-4.5
O 5 -5.0 I
PPm
1
1
I
I
8.5
I
I
I
I
,
/
,
I
PPm
7.5
8.0
T2
3CT
rpn
c ?
8
'0
3
Figure 2. Phase-sensitive NOESY spectrum of CTTTNSRGTTT 9 m M in ( C2H,)sS0. Mixing time was 120 ms; sweep width in F2was 6410 Hz; 512 tl increments were implemented over 2048 points in t2dimension. Data matrix was zero filled to 1024 in Fl and a T / 2 shifted sine-bell window was applied in both dimensions; ( a ) NH-CHJCH, region, the asterisk indicates the daN(i, i 2 ) connectivity between the Thr-2 and Thr-4, ( b ) NH-NH region, and ( c ) CH,-CH, region.
+
A set of distances was obtained (see Table 111) from NOESY data employing the initial rate approximation method, l5 by using the fixed distance of the methylene protons in Asn-5 and Arg-7 as internal calibration. T h e observation of strong d a N ( i, i 1) NOE connectivities over the entire peptide means that the
+
conformational ensemble of the peptide includes a population of extended chain forms, which appear as predominating in the unfolded structures." In addition, d N Ni, ( i 1) and few dnN(i, i 2 ) NOE connectivities are observed they indicate the presence of a pronounced preference for folded conformations. T h e consecutive d"(i, i 1) NOE con-
+
+
+
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
719
:.5
2.0
0
2.5 4
0 3.0
0 8
3
3.5
0 4.0
4.5
Q
I
bJ
@-
0
5.0 PPm PPm
4.5
4.0
3.5
Figure 2.
+
2.5
2.0
(continued from the previous page)
nectivities are as would be expected for a n a- or 310helix2'; however, none of the characteristic d n N ( i , i 3 ) , or d,,$(i, i 3 ) NOE connectivities, diagnostic of a regular helix, were observed. On the other hand, the observed high values of the NH-aH coupling constants, JNH.aH, for well-resolved NH protons in the 1D spectrum (see Table 11),are not in agreement with the expected low values 3 J ~ ~ = 4. Hz, c ychar~ acteristic of a - or 310-helices.'5Nor are the data consistent with the presence of P-sheet elements, where d" connectivities are not usually detected, and 3 J N ~ - a=~ 9 Hz, which is not the present case.
+
3.0
+
2 ) connectivities indicate the T h e d,N(i, i presence of local structures and short-range order. We also notice the presence of d o N ( i, i 1) connectivities, which are commonly observed in 0-turns and in helical structures.22 Turns of two general classes, type I and type 11, are possible (type I11 turns are very similar to type I)23;these two types could not be precisely distinguished by the present experiments. Type I and type I1 turns can, in principle, be distinguished on the basis of NOE connectivities and the 3 J ~ coupling , constants a t positions 2 and 3 of the Unfortunately for flexible
+
720
MOLINARI ET AL.
Table I11 Proton Distances Obtained from NMR Data cys-1
Ha-NH(Thr-2) Ha-NH(Thr-3)
2.24 3.39
HP1-NH(Thr-2)
3.29
3.12
Thr-2 NH-NH(Thr-3) Ha-NH
3.11 2.64
Ha-NH(Thr-3) HP-NH
2.10 2.84
Ha-NH(Thr-4) Hr-NH
3.45 2.70
2.16 2.58
NH-Hn
2.63
3.06 2.72
HP2-NH(Ser-6)
3.08
3.22 2.83
NH-Ha
2.47
Thr-3 NH-NH(Thr-4) NH-HB
2.69 2.61
Ha-NH(Thr-4) NH-Hy Thr-4
HP-NH(Asn-5)
2.71
NH-Ha
2.71 Asn-5
Ha-NH(Ser-6) NH-HB1
2.20 2.83
HP1-NH(Ser-6) NH-HP2 Ser-6
HP1-NH(Arg-7) NH-HP1
3.15 2.83
HP2-NH(Arg-7) NH-HPB Arg-7
Ha-NH(Gly-8)
2.20
NH-H-/l
3.04 Gly-8
NH-NH(Thr-9)
2.82
Ha-NH(Thr-9)
linear peptides in solution, the coupling constants represent a n ensemble average and are of less value than other parameters in distinguishing between classes of 0-turns. A large population is necessary in order that a significantly lower coupling constant is observed, since the value for fully folded turns is 5 H z . Our ~ ~ nmr data slightly favor a type I1 turn, in that strong d o N ( i, i f 1)NOE connectivities are observed, characteristic of type I1 t ~ r n . ~ ~ , ’ ~ Thus nmr data all together seem t o indicate that the @-turnsare one important family of a n ensemble of conformations available to the peptide. T h e peptide then adopts a number of transient local conformations, the turn-like structures, all of which may interconvert rapidly by way of a n extended “random coil” form. In agreement with the results recently reported by Dyson et al., 27 this ensemble of secondary structures could be defined as a “nascent helix.” As reported by D y ~ o n , ’the ~ NOEs for a flexible peptide are a population-weighted average of the
2.32
NOEs in all of the conformational states available t o the peptide. For short linear peptides in solution, the d” ( i , i 1)distance becomes just short enough to give observable NOEs between residues with @ and \k angles in the helix region of (@, \k) space. Further, dNNdoes not vary much over the allowed range of @ and \k angles in this region. Thus differences in intensity of the NH-NH NOEs reflect differences in the population of “helical conformers” a t each residue, once the decreased correlation times a t the ends of the peptide are taken into account. In CTTTNSRGTTT, NOEs of similar intensities are observed throughout the sequence, the most intense peak being observed for the dipolar correlation between T h r - 3 and Thr-4, suggesting that the highest population of structured conformers occurs a t these residues (Thr-4 also exhibits a low-temperature coefficient). Indeed, a preliminary conformational analysis, performed using the SEARCH program of the SYBYL software, indicated a lack of
+
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
convergence t o a unique family of structures, in agreement with the presence of the proposed conformational equilibrium. Experiments in cryoscopic mixtures (DMSO / water) a t low temperatures are under study, with the aim of obtaining a number of distance constraints appropriate for dynamic simulations. 15NSpectroscopy
In view of the importance of structural studies on short synthetic immunogenic peptides, I5N experiments have also been performed. T h e use of 15N, which has a larger chemical shift dispersion than its proton counterpart, has been shown t o be a very useful method for alleviating the problem of spectral overlap. In addition, correlation between 15N chemical shifts and structural features could be of great interest. T o maximize sensitivity, the I5N spectrum is typically measured indirectly, as the F1 dimension of a 2D experiment in which the 'H magnetization is both initially excited and detected. In the experiments shown here, heteronuclear 'H- 15N multiplequantum coherence (HMQC) '*is allowed to evolve during the evolution period tl . Recently, a new experiment has been described that combines heteronuclear correlation and 'H homonuclear NOESY to give 2D spectra with cross peaks between 'H/15N correlation peaks from amide sites and other protons dipolarly coupled to them. The original experiment was performed on uniformly 15N-labeled samples. We report here a study on the applicability of this experiment to our peptide at natural 15Nabundance. T h e sequence employed is depicted in Scheme 1: 13928
90'
180'
90
Scheme 1
T h e HMQC present during the tl evolution period evolves only due to 15Nchemical shifts with respect to tl and consists both of zero- and double-quantum coherences interconverted by the 180" ('H) pulse. After the last 90" ( 15N),which converts the zeroand double-quantum coherences into antiphase detectable proton magnetization, a delay A = 1 /2 J cL5 N. 1H followed ) by a 90" ('H) pulse brings the proton magnetization along the z axis, allowing
721
a n exchange of magnetization between dipolarly coupled protons, during the mixing time 7,. Heteronuclear decoupling during t 2 is mandatory in these kinds of experiments. It is achieved through composite pulse schemes, such as MLEV-4, 29 WALTZ-16,30or GARP-1.14 Their performance, as far as the frequency range of the decoupling is concerned, has been reported.14 The GARP-1 scheme appears to offer the best performance, but the WALTZ-16 scheme is efficient enough when dealing with 15N,which is usually recorded with small sweep width. When the heteronuclear-NOE experiment was performed with a short mixing interval (i.e., 7 , = 10 m s ) , each amide site gave rise to a single 'H/15N cross peak, as shown in Figure 3a. Since it is impossible to distinguish between the overlapping proton resonances of Thr-10 and Thr-11, the assignment of nitrogen resonances, only based on the simple HMQC experiment (Figure 3 a ) , was not complete. For the other two overlapping resonances of Asn-5 and Arg-7, a unique correlation is observed in the heterocorrelated experiment (Figure 3 a ) , indicating that their nitrogen resonances are also degenerate. The analysis of the experiment performed with a longer mixing time (7, = 180 ms; Fig. 3b) reveals additional peaks due to the dipolar correlation between each N H with its a proton. Since the experiment was performed on a 40 m M peptide solution, a NOESY on the same concentrated solution was additionally performed, to check for the proton assignment. From the experiment of Figure 3b it is possible to distinguish between Thr-10 and Thr-11, which correlate with a H s occurring a t different chemical shifts-an indication of the potentiality of this kind of experiment for 15N assignment. All the other 15N assignments were confirmed. T h e analysis of cross sections revealed the presence of cross peaks in the amide region as well, one between the Thr-3 and Thr-4, thus confirming the previous hypothesis of a highest population of structured conformers occurring a t these residues, and a weak one between Thr-4 and Asn-5. I t is apparent that in this experiment the strong intraresidue NOES are observed, while only a couple of NHN H connectivities could be detected from the analysis of cross sections. This deserves some remarks. T h e HMQC sequence belongs t o the class of pulse schemes in which the satellite signals are selected by phase cycling alone3' and is therefore well suited to detect enriched signals. The stringent requirements of stability and dynamic range imposed on the nmr spectrometer when the signals selection is accomplished by phase cycling only, together with
722
MOLINARI ET AL.
105
1
110
i'
I
3
-
45 77
I
1H
ppn
8.0
6.0
7.0
510
410
1H
Figure 3. Phase-sensitive heteronuclear 'H- 15N spectrum of CTTTNSRGTTT obtained by employing the HMQC sequence modified to record NOE effects. The experiment was performed on a natural 15Nabundance, 40 m M in ( C2H3)2SO;sweep width was 5000 and 2000 Hz in F2 and F, , respectively; 94 tl increments, each of 2560 scans, were implemented over 2048 points in t2 dimension. Data matrix was zero filled to 512 in F , and a ?r/2 shifted sine-bell window was applied in both dimensions; ( a ) mixing time 10 ms, and ( b ) mixing time 180 ms.
the incomplete suppression of the main signals from the protons bound t o 14N, decrease the sensitivity of the HMQC scheme when applied to natural abundance samples. In addition, the HMQC exhibits in F1the homonuclear multiplet of its active protons and the effective linewidth will be unfavorable if compared to single quantum coherence experiments, as recently r e p ~ r t e d . ~All " ~these ~ factors are certainly crucial in a sensitive experiment as the detection of small NOE effects in a reverse pulse scheme, and this could explain the lack of the weaker NH-NH cross peaks from the experiment of Figure 3b. It is worth mentioning that, in the described heteronuclear-NOE experiment applied to natural abundance 15N samples, a much higher number of transients, with respect to those necessary to obtain a normal HMQC correlation, is necessary for each tl . As a result, quite long overall acquisition times are required (3-4 days). In spite of this drawback, it is still very advantageous to get 15N spectra with unprohibitive amounts of biological sample. In order to improve the performance of the described method we are presently studying other sequences, based on
the evolution of 15N single quantum coherence, on which pulses for NOE detection are superimposed.
CONCLUSIONS T h e combined use of 'H and 15N high-field nmr spectroscopy provided the complete assignment of both proton and nitrogen resonances for the undecapeptide CTTTNSRGTTT in DMSO solution. In particular, the heteronuclear-NOE reverse experiment offers the possibility of assigning unambiguously the "N spectra, irrespective of overlapping in the proton resonances. 15N data may play a n important role in view of studies on synthetic immunogenic peptides bound to antibodies. Structural data obtained from NOESY experiments agree with the presence of ordered structures in the peptide. T h e observation that this short immunogenic peptide fragment may adopt ordered structures bears significantly on several fundamental issues, including the problem of protein folding.z3x33Stable local structures, which may be different from those in the final folded protein, can be
DERIVATIVE OF A RABIES GLYCOPROTEIN FRAGMENT
recognized by antibodies, and have been postulated as initiation sites in f ~ l d i n g . The ~ ~ -experimental ~~ observations described here may thus link the immunological and theoretical approaches to protein folding, and show that the combined use of synthetic immunogenic fragments of proteins and high-field nmr spectroscopy provides a powerful approach to the identification of local structures of biological relevance. We thank Giulio Zannoni and Fulvia Greco for their expert technical assistance.
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Received July 20, 1990 Accepted December 5, 1990
