'H-NMR Assignments and Conformational Studies of Melanin Concentrating Hormone in Water Using Two-Dimensional NMR * TERRY 0. MATSUNACA, CATHERINE A. CEHRIC, and VICTOR J. HRUBY *' Department of Chemistry, University of Arizona, Tucson, Arizona 85721
Salmon melanin concentrating hormone (MCH) , a heptadecapeptide that stimulates the perinuclear aggregation of melanin granules ( melanosomes) within teleost melanocytes, is a cyclic peptide with the following sequence:
backbone and side-chain protons of MCH in 90% H20/ 10% D20. The assignments permit deductions regarding its secondary structure.
MATERIALS A N D METHODS I Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val MCH was synthesized and isolated as the trifluoroacetate -Tyr-Arg-Pro-Cis-Trp-Glu-Val (TFA) salt? The TFA was exchanged for chloride by passing an aqueous solution thru a diethylaminoethyl Recent experimental evidence suggests that vertebrate (DEAE) Sephadex A-25 anion exchange resin generated animals may possess separate receptors for MCH and its in the C1- form. The product was lyophilized, then reconhormonal counterpart, a-melanocyte stimulating hormone stituted to 7 m M in 90% HzO/lO% 'H20 (Aldrich, low ( a - M S H ).' Despite evidence for separate receptors, exparamagnetic, T I= 48.1 s ) . The pH was adjusted to 4.6 perimental data also suggests that MCH can stimulate an with 1 N HCI. a-MSH-like response in amphibian and reptilian modAll nmr measurements were conducted a t 500.13 MHz e l ~ . *These -~ findings have led some to suggest a structural on a Bruker AM-500 spectrometer. All 2D spectra were or evolutionary link between the two hormones and/or recorded in the pure absorption mode using the time protheir receptors." Recently, the gene encoding for MCH portional phase incremention ( T P P I ) method."." Twohas been isolated from chum salmon liver.6 Also, a large dimensional phase-sensitive NOESY spectra were reamount of sequence homology has been found when comcorded with the pulse sequence to-9O0-t1-9Oo-~,-9O0-t2 ." paring the salmon mRNA to human and rat mRNA seA 90" pulse of 6.5 p s was determined. Within a single tl quences.' value ( 3 p s ) mixing times T , varied from 50 to 500 ms. The question of a structural link between MCH and All NOES were determined and quantitated at a mixing MSH has led us to study the conformation of MCH in time of 200 ms. A 10% variation in the mixing time was solution by nmr spectroscopy. Peptides or linear fragments used to suppress zero-quantum coherence between spins of proteins of this size and magnitude are not as disordered with shift differences greater than 0.1 ppm. The 2D as originally believed, and actually can have a good deal TOCSY were recorded with the pulse sequence to-90"-t,of relatively stable secondary structure? The presence of SL,- (MLEV17) -SL,-t2, where SL, is a 2.5 ms trim pulse, a cyclic disulfide ring in MCH enhances the probability directed along the x axis, to defocus magnetization not of a structurally defined peptide. Using recent advances parallel to the x axis. In lieu of trim pulses, TOCSY exin two-dimensional ( 2D ) methodology including phaseperiments were also performed with a 2 filter before and sensitive nuclear Overhauser enhanced spectroscopy after the mixing time.13In TOCSY experiments, the pulse ( NOESY ) , and phase-sensitive correlated spectroscopy sequence was run in the inverse mode to adjust the 90' (COSY), we have been able to ascertain considerable pulse to between 20 and 28 p s in duration. In addition, conformational information about MCH. the number of MLEV17 phase cycles was adjusted to allow Techniques [such as total correlation spectroscopy between 30 and 100 ms duration for resolution of remote (TOCSY), phase-sensitive NOE, and double quantum filconnectivities. DQF COSY used a t,,-90°-tl-900-A-900-tz tered (DQF) COSY] are used to assign protons of the pulse sequence, where t l , t2, and A are the evolution, detection, and fixed delay periods, respectively. Baseline distortions were corrected by applying linear frequency0 1990 John Wiley & Sons, Inc. dependent phase corrections in the frequency domain.14 CCC 0006-3525/90/13-141291-05 $04.00 The NOESY and DQF COSY spectra were acquired Biopolymers, Vol. 30, 1291-1295 (1990) with a spectral width of 6024 Hz. TOCSY experiments * Presented in part at the 11th American Peptide Symposium, were recorded with a spectral width of 8032 Hz t o observe La Jolla, California, July 9-14, 1989. remote connectivities (i.e., all Arg and Val). Usually 512 To whom reprint requests should be addressed.
'
1291
1292
BIOPOLYMERS, VOL. 30 (1990)
or 700 tl values were acquired. Free induction decays consisted of 2048 data points and a low power preirradiation duration ( t o )of 1.5 s. All data prior to Fourier transformation were multiplied by a sine-bell function phase shifted by 90" or 60", and zero filled to 2048 complex data points in the t2 dimension. Similar zero filling and multiplications were conducted in the tl dimension. Digital resolution allowed a confidence limit to 0.01 ppm. All chemical shifts were assigned relative to our reference standard, 2- (trimethylsily1)propionate 2,2,3,3-d4, sodium salt. All processing of nmr data was performed using the program FTNMR (Hare Research Inc., Woodinville, WA). Approximate distances were obtained from cross-relaxation rates based upon a known fixed distance, from the equationI5
The known distance rkl is the distance between geminal protons of Gly' (1.78 A ) . Calculated distances were then applied as input constraints for DISGEO by classifying an upper limit for each distance. All distances fall into one of three categories; < 2.5, < 3.5, and < 5.0 8, corresponding to strong, medium, and weak NOEs, respectively. An upper limit of 5.0 A for weak NOEs was used because most weak interactions involved side-chain protons that reflect a higher flexibility than for backbone segments.16 Upper limits for distances involving pseudoatoms were
corrected for center averaging as described by Wuthrich et al.17A total of 11independent structures were obtained by changing the random seed number at the beginning of each calculation. An rms difference for all backbone atoms ( H , N, Ccu, C, and 0 ) was determined using a program graciously provided to us by Dr. I. D. Kuntz and John Thomason at the University of California at San Francisco.
RESULTS Assignment of Resonances Spectral assignments of proton resonances were obtained by the sequential resonance assignment m e t h ~ d . ' ~ In,'~ dividual amino acid spin systems were identified primarily by TOCSY spectra and verified by DQF COSY. Figure 1B displays the NH-CH, region of the unsymmetrized phasesensitive NOE spectrum. Assignments were determined initially by first observing the methyl groups of the Val7, Val", and Va1I7. The only other methyl resonance observed was from the Thr', which displayed a resonance downfield to the valine methyls. This is primarily due to the deshielding component imparted by the b-OH group. Using the sequential resonance assignment approach, we can unequivocally assign all the NH-CH, connectivities from T h r 2 to Arg". Once Val7and Val" were assigned, Val17was then assigned by the process of elimination, and subsequent sequential
Figure 1. The 500.13-MHz 'H phase-sensitive NOESY spectrum of MCH. ( A ) Amide backbone region of MCH illustrating NH-NH dipolar couplings. ( B ) The fingerprint region of MCH illustrating the sequential assignments as well as some NOE secondary assignments. Of significance is the i - i 2 interaction between the CH,s of Gly' and the NH of Val"
+
RESEARCH COMMUNICATIONS
assignment from Val'7 to Cys'* was achieved. The CH, and CH, of Asp' were assigned by the NOE from the CH, of Asp' to the NH of Thr'. Pro13was assigned by observing the remaining CH, resonance near the diagonal and determining @, y , and 6 connectivities via the TOCSY experiments. When possible, diastereotopic protons were assigned according to their shifts. Some of the cross peaks due to NH-CH, scalar couplings can also be seen in the NOE spectrum. For the purposes of modeling, we have tentatively made the assumption that the NH-CH, scalartype interactions not observed in the NOESY spectrum may be trans periplanar in proximity to each other. Table I lists the chemical shift assignments for MCH. Once the peptide was completely assigned, we set out to determine the dipolar connectivities by observing the phase-sensitive NOE spectra. To date, we have found 69 NOE assignments (of which about 60% are interresidual) in the resolved spectra. Of particular interest was the observation of a medium range (2.5-3.5 A ) NOE between the diastereotopic CH, protons of Gly8 and the NH proton of Val'' (Figure 1 B ) . Observation of such an i - i 2 CH,,-NH interaction often is an indication of a P-turn. This, coupled to the observation of a strong NOE ( < 2.5 A ) between the Argg and Val" NHs, as well as the interaction between the Gly' and Argg NHs, provides very good evidence for a type I @-turn(Figure 1A) .'' In addition, the diastereotopic nature of the Gly8 CH,s is also suggestive of secondary structure in this region. The amide region of the nmr spectrum is also displayed, which defines the NH-NH interactions between Gly8-Argg,Argg-ValIo,and Val"-Tyr", as well as others. Figure 2A displays a graphic representation of some of the pertinent NOEs.
+
Table I
1293
Distance geometry calculations using the program DISGE021~22 have tentatively identified this reverse turn as a type I @-turn.Figure 2B displays the MCH 11 superimposed structures generated from DISGEO. Of the 11 structures generated, the backbone portion of the molecule confined to the ring appears to vary less than the exocyclic portion of the backbone. This observation is supported by the fact that the rms difference, when compared to the average coordinates, is 2.9 A for the entire molecule, but a much more reasonable 1.7 A when only the average of the 5-14 residues of the ring portion of the molecule is calculated. This appears reasonable, as the component of conformational constraint imparted by the Cys5-Cys l 4 disulfide bridge should restrict the torsional motions of the residues in the ring. A transannular interaction has also been identified between the 2'-6'protons of Tyr" to both the Val7 NH and the Met' CH,. In addition, NOEs to the CH,, of Val'" and the intraresidual NH to the Tyr" aromatic protons have been identified. These NOEs in water are similar to the transannular effect suggested by Raker et a1." in their nmr studies in DMSO-d, of the MCH, 14 fragment of MCH. It appears that the exocyclic moieties do not perturb the ring-spanning effect of the Tyr" .
DISCUSSION Recent have demonstrated that the MCH molecule can also stimulate MSH-like activity in some systems. Because the receptor ( s ) have not been characterized
'H Assignments of MCH as Determined at 500.13 MHz 'H Assignments
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. a
Asp Thr Met Arg
cys Met Val Gly Arg Val Tyr Arg
Pro Cys Trp Glu Val
NH
CH,
CH,
NO a 8.75 8.54 8.53 8.96 8.69 8.65 8.88 8.68 7.66 8.42 8.69
4.38 4.42 4.61 4.71 5.05 4.62 4.14 4.00 (3.71) 4.33 4.34 4.94 4.78 4.35 4.76 4.79 4.41 4.07
2.84 4.20 2.59 1.65 (1.58) 3.01 (2.88) 1.89 1.89
-
8.72 7.75 7.88 7.69
NO: n o t observable.
2.02 (1.76) 2.07 2.86 (2.82) 1.80 2.00 (1.87) 1.28 3.34 2.00 2.13
CH,
1.25 2.06 1.45
CHa
NH,
2.95
7.06
1.60 0.91
3.19
7.19
1.54 (1.53) 1.31
3.12 3.70 (3.45)
7.21
1.65 0.84
1.68 0.89
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BIOPOLYMERS, VOL. 30 (1990)
1
5
15
10
B
A
Figure 2. ( A ) Sequential short-, medium-, and long-range NOES detected in the NOESY spectrum of MCH (mixing time equals 200 ms) . Thickness of the lines indicates the relative intensities of the cross peaks in the NOESY spectrum. ( B ) Backbone conformation of the 5-14 ring for the 11 structures with the NOE input constraints programmed into the DISGEO modeling program (rms deviation = 1.7 A ) .
to date, we have set out to define the conformational and topographical features of the ligand (MCH) in order to get an indication of the features necessary for receptor binding and transduction. Our current observations using nmr spectroscopy have led us to identify a 0-turn region in the molecule as well as a transannular effect of the Tyr" side-chain moiety. The prominent i - i 2 CH,NH NOE of medium proportion as well as the subsequent NH-NH and CH,-CH, interactions between residues 7 and 10 have led us to conclude that a type I 0-turn exists in this region, and this is supported by distance geometry calculations. In addition to a more comprehensive evaluation of these nmr results, experiments are currently being conducted to apply both conformational and topographical constraints via the use of peptide mimetics and constrained analogues to help determine the requirements of the receptor for binding and transduction.
+
The work was funded through grants from the National Science Foundation (DMB-8712133) and the U.S. Public Health Service (DK 17420). The authors would like to express their gratitude to Dr. Paul Gooley, Dr. Kenner Christensen and Dr. Michael Barfield for their nmr technical advice. We also would like to acknowledge the help of Ms. Susan Yamamura and Mr. Constantine Job for their expert advice regarding computer simulation applications. The assistance of Dr. B. Montgomery Pettitt at the University of Houston is also gratefully acknowledged. Terry 0. Matsunaga acknowledges the National Institute on Drug Abuse for the generous support of a NIDA postdoctoral fellowship (DA 05371-02).
REFERENCES 1. Oshima, N., Kasukawa, H., Fujii, R., Wilkes, B. C., Hruby, V. J., Castrucci, A. M. L. & Hadley, M. E. (1985) J . Exp. 2001.235, 175-180. 2. Wilkes, B. C., Hruby, V. J., Castrucci, A. M. L., Sherbrooke, W. C. & Hadley, M. E. (1984) Science 225, 1111-1113. 3. Eberle, A. N., Atherton, E., Dryland, A. & Sheppard, R. C. (1986) J. Chem. SOC.Perkin Trans. 1,361-367. 4. Ide, H. I., Kawazoe, I. & Kawauchi, H. (1985) Gen. Comp. Endocrinol. 58, 486-490. 5. Castrucci, A. M. L., Hadley, M. E., Wilkes, B. C., Zechel, C. & Hruby, V. J. (1987) Life Sci. 40, 845851. 6. Takayama, Y., Wada, C., Kawauchi, H. & Ono, M. (1989) Gene 80,65-73. 7. Nahon, J.-L., Schoepfer, R. & Vale, W. (1989) Nucleic Acid. Res. 17, 3598. 8. Wright, P. C . , Dyson, H. J. & Lerner, R. A. (1988) Biochemistry 27, 7167-7175. 9. Matsunaga, T. O., Castrucci, A. M. L., Hadley, M. E. & Hruby, V. J. (1989) Peptides 10,349-354. 10. Bodenhausen, G., Vold, R. L. & Vold, R. R. ( 1980) J. Magn. Reson. 37, 93-106. 11. Marion, D. & Wuthrich, K. (1983) Biochem. Biophys. Res. Commun. 113,967-974. 12. Jeener, J., Bachmann, P. & Wuthrich, K. (1979) J. Chem. Phys. 7 1,4546-4553. 13. Rance, M. (1987) J. Magn. Reson. 74,557-564. 14. Marion, D. & Bax, A. (1988) J. Magn. Reson. 79, 352-356.
RESEARCH COMMUNICATIONS
15. Olejniczak, E. T., Gampe, R. T., Rockway, T. W. & Fesik, S. W. (1988) Biochemistry 27, 7124-7131. 16. Williamson, M. P., Havel, T. F. & Wuthrich, K. (1985) J . Mol. Biol. 1 8 2 , 295-315. 17. Wuthrich, K., Billeter, M. & Braun, W. (1983) J. Mol. Biol. 169, 949-961. 18. Billeter, M., Braun, W. & Wuthrich, K. (1982) J . Mol. Biol. 1 5 1 , 321-345. 19. Wagner, G., Kumar, A. & Wuthrich, K. (1981) Eur. J. Biochem. 1 1 4 , 375-384. 20. Wuthrich, K. (1986) N M R of Proteins and Nucleic Acids, Wiley, New York, p. 166.
1295
21. Havel, T. & Wuthrich, K. (1984) Bull. Math. Biol. 46,673-698. 22. Havel, T. F., Kuntz, I. D. & Crippen, G. M. (1983) Bull. Math. Biol. 4 5 , 665-695. 23. Baker, B. I., Brown, D. W., Campbell, M. M., Kinsman, R. G., Moss, C. A,, Osguthorpe, P. J., Paul, P. K. C. & White, P. D. (1988) J . Chem. SOC.Chem. Commun. 1543-1545.
Receiued December 4, 1989 Accepted June 14, 1990
Salmon melanin concentrating hormone (MCH) , a heptadecapeptide that stimulates the perinuclear aggregation of melanin granules ( melanosomes) within teleost melanocytes, is a cyclic peptide with the following sequence:
backbone and side-chain protons of MCH in 90% H20/ 10% D20. The assignments permit deductions regarding its secondary structure.
MATERIALS A N D METHODS I Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val MCH was synthesized and isolated as the trifluoroacetate -Tyr-Arg-Pro-Cis-Trp-Glu-Val (TFA) salt? The TFA was exchanged for chloride by passing an aqueous solution thru a diethylaminoethyl Recent experimental evidence suggests that vertebrate (DEAE) Sephadex A-25 anion exchange resin generated animals may possess separate receptors for MCH and its in the C1- form. The product was lyophilized, then reconhormonal counterpart, a-melanocyte stimulating hormone stituted to 7 m M in 90% HzO/lO% 'H20 (Aldrich, low ( a - M S H ).' Despite evidence for separate receptors, exparamagnetic, T I= 48.1 s ) . The pH was adjusted to 4.6 perimental data also suggests that MCH can stimulate an with 1 N HCI. a-MSH-like response in amphibian and reptilian modAll nmr measurements were conducted a t 500.13 MHz e l ~ . *These -~ findings have led some to suggest a structural on a Bruker AM-500 spectrometer. All 2D spectra were or evolutionary link between the two hormones and/or recorded in the pure absorption mode using the time protheir receptors." Recently, the gene encoding for MCH portional phase incremention ( T P P I ) method."." Twohas been isolated from chum salmon liver.6 Also, a large dimensional phase-sensitive NOESY spectra were reamount of sequence homology has been found when comcorded with the pulse sequence to-9O0-t1-9Oo-~,-9O0-t2 ." paring the salmon mRNA to human and rat mRNA seA 90" pulse of 6.5 p s was determined. Within a single tl quences.' value ( 3 p s ) mixing times T , varied from 50 to 500 ms. The question of a structural link between MCH and All NOES were determined and quantitated at a mixing MSH has led us to study the conformation of MCH in time of 200 ms. A 10% variation in the mixing time was solution by nmr spectroscopy. Peptides or linear fragments used to suppress zero-quantum coherence between spins of proteins of this size and magnitude are not as disordered with shift differences greater than 0.1 ppm. The 2D as originally believed, and actually can have a good deal TOCSY were recorded with the pulse sequence to-90"-t,of relatively stable secondary structure? The presence of SL,- (MLEV17) -SL,-t2, where SL, is a 2.5 ms trim pulse, a cyclic disulfide ring in MCH enhances the probability directed along the x axis, to defocus magnetization not of a structurally defined peptide. Using recent advances parallel to the x axis. In lieu of trim pulses, TOCSY exin two-dimensional ( 2D ) methodology including phaseperiments were also performed with a 2 filter before and sensitive nuclear Overhauser enhanced spectroscopy after the mixing time.13In TOCSY experiments, the pulse ( NOESY ) , and phase-sensitive correlated spectroscopy sequence was run in the inverse mode to adjust the 90' (COSY), we have been able to ascertain considerable pulse to between 20 and 28 p s in duration. In addition, conformational information about MCH. the number of MLEV17 phase cycles was adjusted to allow Techniques [such as total correlation spectroscopy between 30 and 100 ms duration for resolution of remote (TOCSY), phase-sensitive NOE, and double quantum filconnectivities. DQF COSY used a t,,-90°-tl-900-A-900-tz tered (DQF) COSY] are used to assign protons of the pulse sequence, where t l , t2, and A are the evolution, detection, and fixed delay periods, respectively. Baseline distortions were corrected by applying linear frequency0 1990 John Wiley & Sons, Inc. dependent phase corrections in the frequency domain.14 CCC 0006-3525/90/13-141291-05 $04.00 The NOESY and DQF COSY spectra were acquired Biopolymers, Vol. 30, 1291-1295 (1990) with a spectral width of 6024 Hz. TOCSY experiments * Presented in part at the 11th American Peptide Symposium, were recorded with a spectral width of 8032 Hz t o observe La Jolla, California, July 9-14, 1989. remote connectivities (i.e., all Arg and Val). Usually 512 To whom reprint requests should be addressed.
'
1291
1292
BIOPOLYMERS, VOL. 30 (1990)
or 700 tl values were acquired. Free induction decays consisted of 2048 data points and a low power preirradiation duration ( t o )of 1.5 s. All data prior to Fourier transformation were multiplied by a sine-bell function phase shifted by 90" or 60", and zero filled to 2048 complex data points in the t2 dimension. Similar zero filling and multiplications were conducted in the tl dimension. Digital resolution allowed a confidence limit to 0.01 ppm. All chemical shifts were assigned relative to our reference standard, 2- (trimethylsily1)propionate 2,2,3,3-d4, sodium salt. All processing of nmr data was performed using the program FTNMR (Hare Research Inc., Woodinville, WA). Approximate distances were obtained from cross-relaxation rates based upon a known fixed distance, from the equationI5
The known distance rkl is the distance between geminal protons of Gly' (1.78 A ) . Calculated distances were then applied as input constraints for DISGEO by classifying an upper limit for each distance. All distances fall into one of three categories; < 2.5, < 3.5, and < 5.0 8, corresponding to strong, medium, and weak NOEs, respectively. An upper limit of 5.0 A for weak NOEs was used because most weak interactions involved side-chain protons that reflect a higher flexibility than for backbone segments.16 Upper limits for distances involving pseudoatoms were
corrected for center averaging as described by Wuthrich et al.17A total of 11independent structures were obtained by changing the random seed number at the beginning of each calculation. An rms difference for all backbone atoms ( H , N, Ccu, C, and 0 ) was determined using a program graciously provided to us by Dr. I. D. Kuntz and John Thomason at the University of California at San Francisco.
RESULTS Assignment of Resonances Spectral assignments of proton resonances were obtained by the sequential resonance assignment m e t h ~ d . ' ~ In,'~ dividual amino acid spin systems were identified primarily by TOCSY spectra and verified by DQF COSY. Figure 1B displays the NH-CH, region of the unsymmetrized phasesensitive NOE spectrum. Assignments were determined initially by first observing the methyl groups of the Val7, Val", and Va1I7. The only other methyl resonance observed was from the Thr', which displayed a resonance downfield to the valine methyls. This is primarily due to the deshielding component imparted by the b-OH group. Using the sequential resonance assignment approach, we can unequivocally assign all the NH-CH, connectivities from T h r 2 to Arg". Once Val7and Val" were assigned, Val17was then assigned by the process of elimination, and subsequent sequential
Figure 1. The 500.13-MHz 'H phase-sensitive NOESY spectrum of MCH. ( A ) Amide backbone region of MCH illustrating NH-NH dipolar couplings. ( B ) The fingerprint region of MCH illustrating the sequential assignments as well as some NOE secondary assignments. Of significance is the i - i 2 interaction between the CH,s of Gly' and the NH of Val"
+
RESEARCH COMMUNICATIONS
assignment from Val'7 to Cys'* was achieved. The CH, and CH, of Asp' were assigned by the NOE from the CH, of Asp' to the NH of Thr'. Pro13was assigned by observing the remaining CH, resonance near the diagonal and determining @, y , and 6 connectivities via the TOCSY experiments. When possible, diastereotopic protons were assigned according to their shifts. Some of the cross peaks due to NH-CH, scalar couplings can also be seen in the NOE spectrum. For the purposes of modeling, we have tentatively made the assumption that the NH-CH, scalartype interactions not observed in the NOESY spectrum may be trans periplanar in proximity to each other. Table I lists the chemical shift assignments for MCH. Once the peptide was completely assigned, we set out to determine the dipolar connectivities by observing the phase-sensitive NOE spectra. To date, we have found 69 NOE assignments (of which about 60% are interresidual) in the resolved spectra. Of particular interest was the observation of a medium range (2.5-3.5 A ) NOE between the diastereotopic CH, protons of Gly8 and the NH proton of Val'' (Figure 1 B ) . Observation of such an i - i 2 CH,,-NH interaction often is an indication of a P-turn. This, coupled to the observation of a strong NOE ( < 2.5 A ) between the Argg and Val" NHs, as well as the interaction between the Gly' and Argg NHs, provides very good evidence for a type I @-turn(Figure 1A) .'' In addition, the diastereotopic nature of the Gly8 CH,s is also suggestive of secondary structure in this region. The amide region of the nmr spectrum is also displayed, which defines the NH-NH interactions between Gly8-Argg,Argg-ValIo,and Val"-Tyr", as well as others. Figure 2A displays a graphic representation of some of the pertinent NOEs.
+
Table I
1293
Distance geometry calculations using the program DISGE021~22 have tentatively identified this reverse turn as a type I @-turn.Figure 2B displays the MCH 11 superimposed structures generated from DISGEO. Of the 11 structures generated, the backbone portion of the molecule confined to the ring appears to vary less than the exocyclic portion of the backbone. This observation is supported by the fact that the rms difference, when compared to the average coordinates, is 2.9 A for the entire molecule, but a much more reasonable 1.7 A when only the average of the 5-14 residues of the ring portion of the molecule is calculated. This appears reasonable, as the component of conformational constraint imparted by the Cys5-Cys l 4 disulfide bridge should restrict the torsional motions of the residues in the ring. A transannular interaction has also been identified between the 2'-6'protons of Tyr" to both the Val7 NH and the Met' CH,. In addition, NOEs to the CH,, of Val'" and the intraresidual NH to the Tyr" aromatic protons have been identified. These NOEs in water are similar to the transannular effect suggested by Raker et a1." in their nmr studies in DMSO-d, of the MCH, 14 fragment of MCH. It appears that the exocyclic moieties do not perturb the ring-spanning effect of the Tyr" .
DISCUSSION Recent have demonstrated that the MCH molecule can also stimulate MSH-like activity in some systems. Because the receptor ( s ) have not been characterized
'H Assignments of MCH as Determined at 500.13 MHz 'H Assignments
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. a
Asp Thr Met Arg
cys Met Val Gly Arg Val Tyr Arg
Pro Cys Trp Glu Val
NH
CH,
CH,
NO a 8.75 8.54 8.53 8.96 8.69 8.65 8.88 8.68 7.66 8.42 8.69
4.38 4.42 4.61 4.71 5.05 4.62 4.14 4.00 (3.71) 4.33 4.34 4.94 4.78 4.35 4.76 4.79 4.41 4.07
2.84 4.20 2.59 1.65 (1.58) 3.01 (2.88) 1.89 1.89
-
8.72 7.75 7.88 7.69
NO: n o t observable.
2.02 (1.76) 2.07 2.86 (2.82) 1.80 2.00 (1.87) 1.28 3.34 2.00 2.13
CH,
1.25 2.06 1.45
CHa
NH,
2.95
7.06
1.60 0.91
3.19
7.19
1.54 (1.53) 1.31
3.12 3.70 (3.45)
7.21
1.65 0.84
1.68 0.89
1294
BIOPOLYMERS, VOL. 30 (1990)
1
5
15
10
B
A
Figure 2. ( A ) Sequential short-, medium-, and long-range NOES detected in the NOESY spectrum of MCH (mixing time equals 200 ms) . Thickness of the lines indicates the relative intensities of the cross peaks in the NOESY spectrum. ( B ) Backbone conformation of the 5-14 ring for the 11 structures with the NOE input constraints programmed into the DISGEO modeling program (rms deviation = 1.7 A ) .
to date, we have set out to define the conformational and topographical features of the ligand (MCH) in order to get an indication of the features necessary for receptor binding and transduction. Our current observations using nmr spectroscopy have led us to identify a 0-turn region in the molecule as well as a transannular effect of the Tyr" side-chain moiety. The prominent i - i 2 CH,NH NOE of medium proportion as well as the subsequent NH-NH and CH,-CH, interactions between residues 7 and 10 have led us to conclude that a type I 0-turn exists in this region, and this is supported by distance geometry calculations. In addition to a more comprehensive evaluation of these nmr results, experiments are currently being conducted to apply both conformational and topographical constraints via the use of peptide mimetics and constrained analogues to help determine the requirements of the receptor for binding and transduction.
+
The work was funded through grants from the National Science Foundation (DMB-8712133) and the U.S. Public Health Service (DK 17420). The authors would like to express their gratitude to Dr. Paul Gooley, Dr. Kenner Christensen and Dr. Michael Barfield for their nmr technical advice. We also would like to acknowledge the help of Ms. Susan Yamamura and Mr. Constantine Job for their expert advice regarding computer simulation applications. The assistance of Dr. B. Montgomery Pettitt at the University of Houston is also gratefully acknowledged. Terry 0. Matsunaga acknowledges the National Institute on Drug Abuse for the generous support of a NIDA postdoctoral fellowship (DA 05371-02).
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RESEARCH COMMUNICATIONS
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Receiued December 4, 1989 Accepted June 14, 1990
