- Email: [email protected]
A Method for Sequential NMR Assignment of1H and13C Resonances of N-Substituted Glycine Peptoids
JOURNAL OF MAGNETIC RESONANCE, ARTICLE NO.
Series B 110, 195–197 (1996)
0030
NOTES A Method for Sequential NMR Assignment of 1H and Resonances of N-Substituted Glycine Peptoids
13
C
ERIN K. BRADLEY Chiron Corporation, Drug Discovery Research, 4560 Horton Street, Emeryville, California 94608 Received July 10, 1995; revised October 30, 1995
Synthesis and screening of diverse chemical libraries for pharmaceutical-lead discovery has been a topic of great interest in recent years. Oligo-N-substituted glycine (NSG) peptoids have been used as a source of chemical diversity, and submonomer synthesis of these libraries has allowed the generation of greater chemical diversity than that available with peptides (1–4). Recently, two trimers, from an NSGpeptoid library of over 3600 compounds, have been identified as specific, nanomolar ligands for two important 7-transmembrane G-protein coupled receptors, a1-andrenergic and m-opiate (5). In view of the pharmaceutical relevance of this new class of compounds, the NMR characterization of NSG peptoids becomes crucial, both for establishing covalent connectivity and for determining the solution and receptor-bound conformations. A prerequisite for achieving these goals is the chemical-shift assignments. A new method is required for NSG-peptoid resonance assignments because of the lack of amide protons on the N-substituted amide bonds. For peptide sequential NMR assignments, spin systems consisting of HN, Ha, Hb1, Hb2, etc., are identified using homonuclear COSY (6, 7) or TOCSY (8) experiments. These homonuclear experiments are adequate for peptide systems, because, for an amino-acid residue, the main-chain protons and most of the side-chain protons are within three bonds of another proton. Thus, most amino-acid monomers form one spin system. However, with NSG peptoids, the side-chain branch point is shifted from the main-chain alpha carbon to the main-chain nitrogen. As a result, each monomer unit has two spin systems, the side-chain branching from the main-chain nitrogen, designated as ‘‘Nxxx,’’ and the two-carbon main-chain unit designated as ‘‘ac’’ (Fig. 1). These subunits are separated by at least four bonds between the side-chain Nxxx–Ha and the main-chain ac–Ha. Described here is a method for assigning the 1H and 13C resonances of NSG peptoids. This method employs both traditional homonuclear sequential assignment techniques (9) and heteronuclear NMR pulse sequences, HMQC (10)
and HMBC (11), for determining the 13C and 1H resonances. The assignments can be completed with this method at natural abundance 13C. However, the initial assignments are considerably facilitated if the NSG peptoid is also available with 13 C label at each main-chain ac–Ca position. Fortunately, these 13C-labeled compounds are straightforward to synthesize with 13C-labeled bromoacetic acid, using the standard submonomer synthesis method (2). The optimal combination of spectra for assignment of NSG peptoids includes natural abundance COSY (or TOCSY), HMQC, and HMBC, and 13C-labeled HMQC and HMBC. Initially, a COSY or TOCSY of the natural-abundance compound is used to establish 1H– 1H connectivities, and a natural-abundance HMQC provides 1H– 13C connectivities. These two experiments establish the proton spin systems and their associated carbon chemical shifts. At this point, the HMQC of the 13C-labeled NSG peptoid is particularly useful to identify unambiguously the main-chain ac–Ca /ac–Ha cross peaks, which occur in the same region at the same peak intensity as the side-chain Nxxx–Ca /Nxxx–Ha cross peaks in a natural-abundance HMQC spectrum. Then an HMBC is performed with the 13C-labeled NSG peptoid, in order to connect the side-chain spin system to the mainchain spin system. The Nxxx–Ha resonances are the only protons connected through three bonds to the 13C-labeled ac–Ca positions (Fig. 1). Thus, a cross peak observed between a main-chain ac–Ca and a side-chain Nxxx–Ha in the 13C-labeled HMBC experiment unambiguously connects the two spin systems and identifies them as a monomer unit. This step is general and can be performed with a naturalabundance HMBC, but the ac–Ca /Nxxx–Ha cross peaks must then be distinguished from the other three-bond coupling of Nxxx–Ca to ac–Ha. In peptides, the sequential connection of spin systems is usually performed using 1H– 1H NOESY (12) cross peaks from the amide protons of one residue to the amide, alpha, or beta protons of the next residue (11). However, with
195
m4982$7329
02-01-96 09:53:47
magas
1064-1866/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AP: Mag Res
196
NOTES
sequential connectivities can be followed along the main chain, 1 ac–Ha to 1 ac–CO to 2 ac–Ha to 2 ac–CO to 3 ac–Ha to 3 ac–CO, etc., as indicated in Fig. 1. The tracing of the main-chain 13C and 1H resonances is illustrated for the trimer peptoid, CHIR-2279 (5) (Fig. 2) in the naturalabundance HMBC spectra shown in Fig. 3. This experiment also provides connectivities from side-chain Nxxx–Ha. As a result, there are two sequential connections that can be used to verify the final sequential assignments (Fig. 1). A similar method of sequential assignments has been successfully used for peptides (13, 14) as well as for proteins (15–18). The HMBC experiment is used to make sequential connections in both cases. At natural abundance, the 13C HMBC has been used to connect amide and alpha proton resonances to the carbonyl carbon resonance of the same amino-acid residue and to connect that carbonyl-carbon resonance to the following residue amide and/or alpha proton chemical shifts (13, 14, 18). When isotopic labeling is possible, connections can be made with 15N or 13C HMBC from beta protons to the main-chain nitrogen or carbonyl carbon (15–17). In both peptides and larger proteins, the use of NOESY cross peaks for sequential assignments can lead to ambiguous results, so these direct through-bond connections are much more desirable. This is even truer for NSG peptoids, where it is necessary to connect both the side chain and main chain unambiguously. In some structural states, it is possible for a main-chain ac–Ha to have NOESY cross FIG. 1. (Top) A nomenclature, similar to that established by Wu¨thrich et al. (9) for peptides and proteins, has been adopted for NSG-peptoid nuclei. However, in order to accommodate the chemistry and avoid confusion in naming of the two carbons attached to the main-chain amide nitrogen, the polymer chain is divided into submonomer units. As a result, two pieces (submonomers) are associated with each residue (monomer) number. For example, 1 Nxxx and 1 ac make up residue 1. The acetyl submonomer (ac) consists of atoms Ca, Ha1, Ha2, C, and O. The amine submonomer (Nxxx) varies, depending on the side chain. As an example, the Nhtyr submonomer (Fig. 2) consists of atoms N, Ca, Ha1, Ha2, Cb, Hb1, Hb2, Cg, Cd1, Hd1, Cd2, Hd2, Ce1, He1, Ce2, He2, Cz , Oz , and Hz . (Middle) Intramonomer connections (e.g., 1 Nxxx to 1 ac) are established using the heteronuclear three-bond coupling of Nxxx–Ha to ac–Ca. The dashed arrows indicate cross peaks which connect side-chain spin systems to the main-chain spin system in the HMBC of an NSG-peptoid 13C-labeled at each ac–Ca position (designated by an asterisk). (Bottom) Intermonomer sequential connections are established using heteronuclear two- or threebond couplings of main-chain carbonyl carbons (ac–CO) to side-chain and main-chain protons (Nxxx–Ha and ac–Ha ). The dashed arrows indicate cross peaks found in a natural-abundance HMBC, which are used to trace sequential connectivities.
NSG peptoids, the amide proton is missing. So the naturalabundance HMBC spectrum of the NSG peptoid is used to make the main-chain sequential connections. The HMBC spectra contain cross peaks between main-chain carbonyl carbons (ac–CO) and the main-chain ac–Ha of both the same monomer and of the following monomer. Thus, the
m4982$7329
02-01-96 09:53:47
magas
FIG. 2. CHIR-2279 (5), Nhtyr–ac–Nbiph–ac–Nhphe–ac–NH2 , is shown with amine side-chain and main-chain segments indicated. The NSG peptoid was synthesized using the submonomer method (2) and purified with reverse-phase HPLC, using a H2O/acetonitrile/TFA gradient system. HPLC fractions were lyophilized from acetic acid ( d4).
AP: Mag Res
197
NOTES
tool in analysis of this pharmaceutically important class of compounds. ACKNOWLEDGMENTS The author is grateful to Dr. G. Montelione, Dr. R. Simon, Dr. R. Guiles, and Dr. J. Davis for helpful discussion and encouragement, and to J. Kerr for synthesis of 13C-labeled CHIR-2279.
REFERENCES
FIG. 3. A natural-abundance HMBC (11) spectrum of CHIR-2279 (Fig. 2). The carbonyl-carbon/alpha-proton region is shown with assignments and sequential connections indicated for both a major and a minor conformer. There are three Ha cross peaks at the 2 ac–CO carbon frequency (168 ppm). In addition to the two main-chain cross peaks, 2 ac–Ha (3.85 ppm) and 3 ac–Ha (4.08 ppm), there is also a cross peak from the sidechain 3 Nhphe–Ha (3.60 ppm). This provides a convenient method for verifying side-chain assignments, since the side-chain Nxxx–Ha (if present) will also be within three bonds of the preceding carbonyl carbon. Note that the 2 Nbiph side chain (Fig. 2) does not have an Ha and thus does not have the corresponding cross peak at the 1 ac–CO carbon frequency. The NMR sample was made as an approximately 10 m M peptoid solution in 25% acetonitrile (d3) and 75% D2O, pH 7. The HMBC spectrum was taken at 107C on a Varian Unity 300 with J Å 9 Hz.
peaks to protons other than those of its sequential neighbor or its own side chain. In conclusion, a straightforward method has been described for the sequential assignments of NSG peptoids. It can be employed either at natural abundance or with simple isotopic labeling at the main-chain ac–Ca. In this laboratory, the method is used routinely to assign a diverse set of NSGpeptoid trimers originating from combinatorial libraries (1– 5). In addition to assignment for the diverse set of side chains, the HMBC has proved robust enough in combination with a ROESY to fully assign all four main-chain geometries of NSG trimers where cis and trans geometries are equally populated at both main-chain amide bonds. This method provides unambiguous connectivities along the main chain of these polymeric molecules and should prove a useful
m4982$7329
02-01-96 09:53:47
magas
1. R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A. Jewell, S. Banville, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, D. C. Spellmeyer, R. Tan, A. D. Frankel, D. V. Santi, F. E. Cohen, and P. A. Bartlett, Proc. Natl. Acad. Sci. USA 89, 9367–9371 (1992). 2. R. N. Zuckermann, J. M. Kerr, S. B. H. Kent, and W. H. Moos, J. Am. Chem. Soc. 114, 10,646–10,647 (1992). 3. R. J. Simon, E. J. Martin, S. M. Miller, R. N. Zuckermann, J. M. Blaney, and W. H. Moos, in ‘‘Techniques in Protein Chemistry V’’ (J. W. Crab, Ed.), pp. 533–539, Academic Press, San Diego, 1994. 4. E. J. Martin, J. M. Blaney, M. A. Siani, D. C. Spellmeyer, A. K. Wong, and W. H. Moos, J. Med. Chem. 38, 1431–1436 (1995). 5. R. N. Zuckermann, E. J. Martin, D. C. Spellmeyer, G. B. Stauber, K. R. Shoemaker, J. M. Kerr, G. M. Figliozzi, D. A. Goff, M. A. Siani, R. J. Simon, S. C. Banville, E. G. Brown, L. Wang, L. S. Richter, and W. H. Moos, J. Med. Chem. 37, 2678–2685 (1994). 6. D. Marion and K. Wu¨thrich, Biochem. Biophys. Res. Commun. 113, 967–974 (1983). 7. M. Rance, O. W. Sørensen, G. Bodenhausen, G. Wagner, R. R. Ernst, and K. Wu¨thrich, Biochem. Biophys. Res. Commun. 117, 479–485 (1983). 8. A. A. Bothner-By, R. L. Stephens, J. Lee, C. D. Warren, and R. L. Jeanloz, J. Am. Chem. Soc. 106, 811–813 (1984). 9. K. Wu¨thrich, G. Wider, G. Wagner, and W. Braun, J. Mol. Biol. 155, 311–319 (1982). 10. L. Mueller, J. Am. Chem. Soc. 101, 4481–4484 (1979). 11. A. Bax and M. F. Summers, J. Am. Chem. Soc. 108, 2093–2094 (1986). 12. J. Jeener, B. H. Meier, P. Bachman, and R. R. Ernst, J. Chem. Phys. 71, 4546–4553 (1979). 13. W. Bermel, C. Griesinger, H. Kessler, and K. Wagner, Magn. Reson. Chem. 25, 325–326 (1987). 14. M. Hofmann, M. Gehrke, W. Bermel, and H. Kessler, Magn. Reson. Chem. 27, 877–886 (1989). 15. A. Bax, S. W. Sparks, and D. A. Torchia, J. Am. Chem. Soc. 110, 7926–7927 (1988). 16. G. M. Clore, A. Bax, P. Wingfield, and A. M. Gronenborn, FEBS Lett. 238, 17–21 (1988). 17. M. Ikura, M. Krinks, D. A. Torchia, and A. Bax, FEBS Lett. 266, 155–158 (1990). 18. P. E. Hansen, Biochemistry 30, 10,457–10,466 (1991).
AP: Mag Res
Series B 110, 195–197 (1996)
0030
NOTES A Method for Sequential NMR Assignment of 1H and Resonances of N-Substituted Glycine Peptoids
13
C
ERIN K. BRADLEY Chiron Corporation, Drug Discovery Research, 4560 Horton Street, Emeryville, California 94608 Received July 10, 1995; revised October 30, 1995
Synthesis and screening of diverse chemical libraries for pharmaceutical-lead discovery has been a topic of great interest in recent years. Oligo-N-substituted glycine (NSG) peptoids have been used as a source of chemical diversity, and submonomer synthesis of these libraries has allowed the generation of greater chemical diversity than that available with peptides (1–4). Recently, two trimers, from an NSGpeptoid library of over 3600 compounds, have been identified as specific, nanomolar ligands for two important 7-transmembrane G-protein coupled receptors, a1-andrenergic and m-opiate (5). In view of the pharmaceutical relevance of this new class of compounds, the NMR characterization of NSG peptoids becomes crucial, both for establishing covalent connectivity and for determining the solution and receptor-bound conformations. A prerequisite for achieving these goals is the chemical-shift assignments. A new method is required for NSG-peptoid resonance assignments because of the lack of amide protons on the N-substituted amide bonds. For peptide sequential NMR assignments, spin systems consisting of HN, Ha, Hb1, Hb2, etc., are identified using homonuclear COSY (6, 7) or TOCSY (8) experiments. These homonuclear experiments are adequate for peptide systems, because, for an amino-acid residue, the main-chain protons and most of the side-chain protons are within three bonds of another proton. Thus, most amino-acid monomers form one spin system. However, with NSG peptoids, the side-chain branch point is shifted from the main-chain alpha carbon to the main-chain nitrogen. As a result, each monomer unit has two spin systems, the side-chain branching from the main-chain nitrogen, designated as ‘‘Nxxx,’’ and the two-carbon main-chain unit designated as ‘‘ac’’ (Fig. 1). These subunits are separated by at least four bonds between the side-chain Nxxx–Ha and the main-chain ac–Ha. Described here is a method for assigning the 1H and 13C resonances of NSG peptoids. This method employs both traditional homonuclear sequential assignment techniques (9) and heteronuclear NMR pulse sequences, HMQC (10)
and HMBC (11), for determining the 13C and 1H resonances. The assignments can be completed with this method at natural abundance 13C. However, the initial assignments are considerably facilitated if the NSG peptoid is also available with 13 C label at each main-chain ac–Ca position. Fortunately, these 13C-labeled compounds are straightforward to synthesize with 13C-labeled bromoacetic acid, using the standard submonomer synthesis method (2). The optimal combination of spectra for assignment of NSG peptoids includes natural abundance COSY (or TOCSY), HMQC, and HMBC, and 13C-labeled HMQC and HMBC. Initially, a COSY or TOCSY of the natural-abundance compound is used to establish 1H– 1H connectivities, and a natural-abundance HMQC provides 1H– 13C connectivities. These two experiments establish the proton spin systems and their associated carbon chemical shifts. At this point, the HMQC of the 13C-labeled NSG peptoid is particularly useful to identify unambiguously the main-chain ac–Ca /ac–Ha cross peaks, which occur in the same region at the same peak intensity as the side-chain Nxxx–Ca /Nxxx–Ha cross peaks in a natural-abundance HMQC spectrum. Then an HMBC is performed with the 13C-labeled NSG peptoid, in order to connect the side-chain spin system to the mainchain spin system. The Nxxx–Ha resonances are the only protons connected through three bonds to the 13C-labeled ac–Ca positions (Fig. 1). Thus, a cross peak observed between a main-chain ac–Ca and a side-chain Nxxx–Ha in the 13C-labeled HMBC experiment unambiguously connects the two spin systems and identifies them as a monomer unit. This step is general and can be performed with a naturalabundance HMBC, but the ac–Ca /Nxxx–Ha cross peaks must then be distinguished from the other three-bond coupling of Nxxx–Ca to ac–Ha. In peptides, the sequential connection of spin systems is usually performed using 1H– 1H NOESY (12) cross peaks from the amide protons of one residue to the amide, alpha, or beta protons of the next residue (11). However, with
195
m4982$7329
02-01-96 09:53:47
magas
1064-1866/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AP: Mag Res
196
NOTES
sequential connectivities can be followed along the main chain, 1 ac–Ha to 1 ac–CO to 2 ac–Ha to 2 ac–CO to 3 ac–Ha to 3 ac–CO, etc., as indicated in Fig. 1. The tracing of the main-chain 13C and 1H resonances is illustrated for the trimer peptoid, CHIR-2279 (5) (Fig. 2) in the naturalabundance HMBC spectra shown in Fig. 3. This experiment also provides connectivities from side-chain Nxxx–Ha. As a result, there are two sequential connections that can be used to verify the final sequential assignments (Fig. 1). A similar method of sequential assignments has been successfully used for peptides (13, 14) as well as for proteins (15–18). The HMBC experiment is used to make sequential connections in both cases. At natural abundance, the 13C HMBC has been used to connect amide and alpha proton resonances to the carbonyl carbon resonance of the same amino-acid residue and to connect that carbonyl-carbon resonance to the following residue amide and/or alpha proton chemical shifts (13, 14, 18). When isotopic labeling is possible, connections can be made with 15N or 13C HMBC from beta protons to the main-chain nitrogen or carbonyl carbon (15–17). In both peptides and larger proteins, the use of NOESY cross peaks for sequential assignments can lead to ambiguous results, so these direct through-bond connections are much more desirable. This is even truer for NSG peptoids, where it is necessary to connect both the side chain and main chain unambiguously. In some structural states, it is possible for a main-chain ac–Ha to have NOESY cross FIG. 1. (Top) A nomenclature, similar to that established by Wu¨thrich et al. (9) for peptides and proteins, has been adopted for NSG-peptoid nuclei. However, in order to accommodate the chemistry and avoid confusion in naming of the two carbons attached to the main-chain amide nitrogen, the polymer chain is divided into submonomer units. As a result, two pieces (submonomers) are associated with each residue (monomer) number. For example, 1 Nxxx and 1 ac make up residue 1. The acetyl submonomer (ac) consists of atoms Ca, Ha1, Ha2, C, and O. The amine submonomer (Nxxx) varies, depending on the side chain. As an example, the Nhtyr submonomer (Fig. 2) consists of atoms N, Ca, Ha1, Ha2, Cb, Hb1, Hb2, Cg, Cd1, Hd1, Cd2, Hd2, Ce1, He1, Ce2, He2, Cz , Oz , and Hz . (Middle) Intramonomer connections (e.g., 1 Nxxx to 1 ac) are established using the heteronuclear three-bond coupling of Nxxx–Ha to ac–Ca. The dashed arrows indicate cross peaks which connect side-chain spin systems to the main-chain spin system in the HMBC of an NSG-peptoid 13C-labeled at each ac–Ca position (designated by an asterisk). (Bottom) Intermonomer sequential connections are established using heteronuclear two- or threebond couplings of main-chain carbonyl carbons (ac–CO) to side-chain and main-chain protons (Nxxx–Ha and ac–Ha ). The dashed arrows indicate cross peaks found in a natural-abundance HMBC, which are used to trace sequential connectivities.
NSG peptoids, the amide proton is missing. So the naturalabundance HMBC spectrum of the NSG peptoid is used to make the main-chain sequential connections. The HMBC spectra contain cross peaks between main-chain carbonyl carbons (ac–CO) and the main-chain ac–Ha of both the same monomer and of the following monomer. Thus, the
m4982$7329
02-01-96 09:53:47
magas
FIG. 2. CHIR-2279 (5), Nhtyr–ac–Nbiph–ac–Nhphe–ac–NH2 , is shown with amine side-chain and main-chain segments indicated. The NSG peptoid was synthesized using the submonomer method (2) and purified with reverse-phase HPLC, using a H2O/acetonitrile/TFA gradient system. HPLC fractions were lyophilized from acetic acid ( d4).
AP: Mag Res
197
NOTES
tool in analysis of this pharmaceutically important class of compounds. ACKNOWLEDGMENTS The author is grateful to Dr. G. Montelione, Dr. R. Simon, Dr. R. Guiles, and Dr. J. Davis for helpful discussion and encouragement, and to J. Kerr for synthesis of 13C-labeled CHIR-2279.
REFERENCES
FIG. 3. A natural-abundance HMBC (11) spectrum of CHIR-2279 (Fig. 2). The carbonyl-carbon/alpha-proton region is shown with assignments and sequential connections indicated for both a major and a minor conformer. There are three Ha cross peaks at the 2 ac–CO carbon frequency (168 ppm). In addition to the two main-chain cross peaks, 2 ac–Ha (3.85 ppm) and 3 ac–Ha (4.08 ppm), there is also a cross peak from the sidechain 3 Nhphe–Ha (3.60 ppm). This provides a convenient method for verifying side-chain assignments, since the side-chain Nxxx–Ha (if present) will also be within three bonds of the preceding carbonyl carbon. Note that the 2 Nbiph side chain (Fig. 2) does not have an Ha and thus does not have the corresponding cross peak at the 1 ac–CO carbon frequency. The NMR sample was made as an approximately 10 m M peptoid solution in 25% acetonitrile (d3) and 75% D2O, pH 7. The HMBC spectrum was taken at 107C on a Varian Unity 300 with J Å 9 Hz.
peaks to protons other than those of its sequential neighbor or its own side chain. In conclusion, a straightforward method has been described for the sequential assignments of NSG peptoids. It can be employed either at natural abundance or with simple isotopic labeling at the main-chain ac–Ca. In this laboratory, the method is used routinely to assign a diverse set of NSGpeptoid trimers originating from combinatorial libraries (1– 5). In addition to assignment for the diverse set of side chains, the HMBC has proved robust enough in combination with a ROESY to fully assign all four main-chain geometries of NSG trimers where cis and trans geometries are equally populated at both main-chain amide bonds. This method provides unambiguous connectivities along the main chain of these polymeric molecules and should prove a useful
m4982$7329
02-01-96 09:53:47
magas
1. R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A. Jewell, S. Banville, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, D. C. Spellmeyer, R. Tan, A. D. Frankel, D. V. Santi, F. E. Cohen, and P. A. Bartlett, Proc. Natl. Acad. Sci. USA 89, 9367–9371 (1992). 2. R. N. Zuckermann, J. M. Kerr, S. B. H. Kent, and W. H. Moos, J. Am. Chem. Soc. 114, 10,646–10,647 (1992). 3. R. J. Simon, E. J. Martin, S. M. Miller, R. N. Zuckermann, J. M. Blaney, and W. H. Moos, in ‘‘Techniques in Protein Chemistry V’’ (J. W. Crab, Ed.), pp. 533–539, Academic Press, San Diego, 1994. 4. E. J. Martin, J. M. Blaney, M. A. Siani, D. C. Spellmeyer, A. K. Wong, and W. H. Moos, J. Med. Chem. 38, 1431–1436 (1995). 5. R. N. Zuckermann, E. J. Martin, D. C. Spellmeyer, G. B. Stauber, K. R. Shoemaker, J. M. Kerr, G. M. Figliozzi, D. A. Goff, M. A. Siani, R. J. Simon, S. C. Banville, E. G. Brown, L. Wang, L. S. Richter, and W. H. Moos, J. Med. Chem. 37, 2678–2685 (1994). 6. D. Marion and K. Wu¨thrich, Biochem. Biophys. Res. Commun. 113, 967–974 (1983). 7. M. Rance, O. W. Sørensen, G. Bodenhausen, G. Wagner, R. R. Ernst, and K. Wu¨thrich, Biochem. Biophys. Res. Commun. 117, 479–485 (1983). 8. A. A. Bothner-By, R. L. Stephens, J. Lee, C. D. Warren, and R. L. Jeanloz, J. Am. Chem. Soc. 106, 811–813 (1984). 9. K. Wu¨thrich, G. Wider, G. Wagner, and W. Braun, J. Mol. Biol. 155, 311–319 (1982). 10. L. Mueller, J. Am. Chem. Soc. 101, 4481–4484 (1979). 11. A. Bax and M. F. Summers, J. Am. Chem. Soc. 108, 2093–2094 (1986). 12. J. Jeener, B. H. Meier, P. Bachman, and R. R. Ernst, J. Chem. Phys. 71, 4546–4553 (1979). 13. W. Bermel, C. Griesinger, H. Kessler, and K. Wagner, Magn. Reson. Chem. 25, 325–326 (1987). 14. M. Hofmann, M. Gehrke, W. Bermel, and H. Kessler, Magn. Reson. Chem. 27, 877–886 (1989). 15. A. Bax, S. W. Sparks, and D. A. Torchia, J. Am. Chem. Soc. 110, 7926–7927 (1988). 16. G. M. Clore, A. Bax, P. Wingfield, and A. M. Gronenborn, FEBS Lett. 238, 17–21 (1988). 17. M. Ikura, M. Krinks, D. A. Torchia, and A. Bax, FEBS Lett. 266, 155–158 (1990). 18. P. E. Hansen, Biochemistry 30, 10,457–10,466 (1991).
AP: Mag Res