Molecular Modeling - American Chemical Society

Chapter 3

Determination of Solution Conformation of Receptor-Bound Ligands by N M R Spectroscopy A Transferred Nuclear-Overhauser-Effect Study of Cyclophilin and a Model Substrate 1

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Downloaded by CORNELL UNIV on August 13, 2016 | http://pubs.acs.org Publication Date: December 14, 1994 | doi: 10.1021/bk-1994-0576.ch003

L. T. Kakalis and I. M . Armitage 1

2

Departments of Pharmacology and Diagnostic Radiology, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208066, New Haven, CT 06520-8066

The receptor-bound conformation of weakly binding ligands may be determined from Transferred N O E (TRNOE) measurements. Cyclophilin (CyP) is the receptor of the immunosuppressant cyclosporin A (CsA) and a peptidyl prolyl isomerase (PPIase) that catalyzes the cis-trans isomerism of X -Pro peptide bonds via an unspecified mechanism. The conformation of substrates in the CyP binding site would provide insights into the enzymatic catalytic mechanism. Transferred N O E measurements indicate that the predominantely trans unbound model substrate suc-AAPF -pNAadopts a cis conformation when bound to CyP. Interactions between ligands and macromolecules are fundamental processes for recognition, catalysis and regulation in biology. The structure of the ligand complexed macromolecules can sometimes be determined from either x-ray crystallography in the solid state ( i , 2) or NMR spectroscopy in solution (3, 4). NMR approaches to the study of large macromolecular complexes require the use of methods for the simplification of the congested proton spectra in order to facilitate spectral analysis (4-6). In the case of strongly binding ligands, isotope-editing techniques permit the observation of only those protons that are scalar or dipolar coupled to the isotopically labeled nuclei of the ligand (7). Another approach involves difference spectroscopy of two receptor-ligand complexes prepared with either protonated or deuterated ligand (5, 6). In the case of recombinant proteins, a third approach relies on protein perdeuteration and the structure determination of the deuterated receptor-bound protonated ligand by conventional NMR (8). Central to any structure determination by NMR is the nuclear Overhauser effect (NOE) and its quantification. The Transferred N O E (TRNOE) is an extension of N O E measurements to 0097-6156/94/0576-0029$08.00/0 © 1994 American Chemical Society Kumosinski and Liebman; Molecular Modeling ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

MOLECULAR MODELING

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molecules undergoing facile chemical exchange between a free and a bound state and is ideally suited to studies of weakly binding ligands. The representative, rather than comprehensive, list of TRNOE applications in Table I is indicative of the usefulness of the method. It should be noted that in many of these studies, the large molecular size of the macromoleculeligand complex precludes its straightforward structural investigation with state-of-the-art multidimensional NMR methods.


The TRNOE Principle. The magnitude of the NOE, defined as the fractional change in the intensity of an N M R resonance upon the rf irradiation of another, is proportional to the cross-relaxation rate σ between the corresponding nuclei as represented by equation 1: (1)

The dependence of σ on the internuclear distance r is the basis of the use of NOE in structure determination whereas the dependence of σ on the correlation time T is reflected in the sensitivity of NOE to molecular size and motion. The TRNOE is an extension of N O E to systems in chemical exchange and, following its initial observation (31), has been systematically treated by Clore and Gronenborn (76, 77). It relies on the transfer of cross-relaxation information between two nuclei of the bound ligand to the free ligand resonances via chemical exchange. In the free state, the ligand is characterized by short correlation times (Tc ~ 100 ps) being either at the extreme narrowing limit (co^ic « 1) where the NOEs are small and positive or at its boundary ( ω τ ^ - 1) where the NOEs approach zero. When bound, the ligand's correlation time becomes that of the macromolecule/receptor (ic ~ 10 ns or larger) and it is thus at the spin diffusion limit ( c o ^ i c » 1) where the NOEs are large and negative. In the event of fast chemical exchange, the observed ligand cross-relaxation is the population-weighed average of cross-relaxation values in the free (F) and the bound (B) states c

2

2

2

tfobs = PFcnF + PBcyB

(2)

and negative bound-state NOEs are transferred to the free or exchangeaveraged ligand resonances where they can be easily measured. These negative NOEs, by virtue of their large magnitude, dominate the observed NOEs thus identifying protons spatially close (< 5 Â) in the bound ligand conformation.

Kumosinski and Liebman; Molecular Modeling ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

3.

KAKALIS & ARMTTAGE

Table I.

Solution Conformation of Ligands

TRNOE Studies of Macromolecule-Ligand Complexes

Macromolecule (kDa)

Ligand

Reft.

CMP-KDO Synthetase (97.5) Hemoglobin (67) Aspartate Transcarbamylase (300) EPSP Synthetase (46.5) Dihydrofolate Reductase (18)

Inhibitors ATP, GTP ATP, CTP, ITP Substrate/Product NADP+, thio-NADP+, Inhibitors cAMP, cGMP, Analogues NAD+NADP+, NADPH Substrate ADP, ATP ATP, ATP Analogue Ile, Val Oligopeptide Substrates Peptide (oxytocin) Fibrinogen, Hirudin and Platelet Receptor Peptides Peptide Inhibitor Troponin I Peptides Peptides Disaccharides Prostaglandins Opiates Peptide Antigens Lipid Antigens Carbohydrate Antigen Substrate AZT, Τ Triphosphates DNA Undecamer Codon, Wobble Codon Substrates, Templates Substrate Analogues Acetylcholine G Protein Peptide Peptide (mastoparan) Peptide (mastoparan) Mating Factor Peptide Drug (chlorpromazine) Enkephalin Analogues

6 10 11 12 13, 14

cAMP Receptor Protein (45) Downloaded by CORNELL UNIV on August 13, 2016 | http://pubs.acs.org Publication Date: December 14, 1994 | doi: 10.1021/bk-1994-0576.ch003

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Dehydrogenases (80-336) Peroxidase (42) Kinases (80-230) Methionyl-tRNA Synthetase (66) Isoleucyl-tRNA Synthetase Elastase (26) Neurophysin (22) Thrombin (36) Ribonucleotide Reductase (171) Troponin C (18.5) Molecular Chaperones (60, 70) Ricin Β (34) BSA (66) Antibody Fragment (50) Antibody Fragments (25, 50) Antibody Fragments Monoclonal Antibody (150) Catalytic Antibody (50) HTV Reverse Transcriptase (117) D N A Binding Protein (75) tRNAAsp (20) D N A Polymerase I Fragment (68) Phospholipase A2 (30) Acetylcholine Receptor Rhodopsin G Protein (40) Phospholipid Vesicles Phospholipid Vesicles Phospholipid Vesicles Phospholipid Bilayers


15 16-21 22 23-26 26, 27 28 29, 30 31-34 35-40 41 42, 43 44, 45 46,47 21,48 49, 50 51-56 57 58 59 60 61 62 63, 64 65 66, 67 68 69 70-72 71,73 74 75

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MOLECULAR MODELING

The observation of TRNOEs is subject to the condition: I PfiCTB I » IPFOFI

(3)

so that aobs~PB10RlF

(5)

0


fast exchange requires

which normally translates into k f f > 10 s~l, R l F being the free ligand longitudinal relaxation rate. Assuming a diffusion-controlled kon ~ 10** M " l s"l, one would expect substantial TRNOEs for M L dissociation constants between 0.1 μ Μ to 1 m M . For weaker binding, the bound population fraction Ρ β is too low and the bound state contributes insignificandy to the overall relaxation (condition 3 breaks down). For stronger binding, the exchange is no longer fast (condition 5 breaks down) and information regarding the bound state conformation is not transferred to the free ligand resonances. 0

Experimental Aspects of T R N O E Measurements. The experiments for TRNOE measurements are no different from those routinely used for regular NOE measurements, i.e. the ID steady state/truncated driven NOE experiment and the transient NOE experiment in its ID or 2D (NOESY) version (9). The former is generally more sensitive but i l l suited for quantitative distance determinations whereas the latter is less susceptible to spin diffusion (48), i.e. the spread of magnetization throughout the molecular complex with concomitant loss in structural information. A significant difference from regular NOEs is the slower growth of TRNOEs, whose time-development is proportional to Ρ β σ β rather than simply σ β , thus necessitating longer irradiation, development and mixing times. Specific experimental and methodological aspects of TRNOE measurements have been previously discussed (78, 79). Ratios of ligand to macromolecule typically range between 10 and 30, depending on the dissociation constant and also the size of the protein since the TRNOE magnitude increases with the protein size. A useful variable is the population ratio PF/PB which is related to the association constant and total concentrations of M and L (eq. 4). Provided condition 3 is satisfied, one strives to use as high a PF/PB ratio as possible for maximal sensitivity and



3. KAKALIS & ARMITAGE


33

minimal spin diffusion. Temperature is another important experimental factor. For certain ligand-macromolecule systems, data acquisition at the highest temperature allowed by the sample's thermal stability may be required in order to satisfy the fast exchange condition (58). Alternatively, the dissociation rate koff may be increased by a suitable modification of the ligand (53). A complete treatise of variables such as mixing time, bound correlation time/protein size and fraction of bound ligand Ρ β in NOESY measurements (80) indicates that TRNOE-derived distances are most accurate for low M to L ratios (with Ρ β being 5% or less) that minimize spin diffusion and reasonable mixing times that provide good signal-tonoise. Other points that affect the development of TRNOEs include the presence of internal mobility of the bound ligand (33, 81), finite boundfree exchange rates (32, 82) and protein indirect relaxation effects (87). Rigorous analysis of TRNOE data requires a relaxation matrix treatment that takes into account the chemical exchange rates. Fast chemical exchange does not present any complications (79, 83, 84). However, in the event of intermediate chemical exchange, the independently determined exchange rates must be included in the calculations (79, 82, 84). These may be obtained from relaxation and saturation transfer measurements (22,58,85, 86). Technical developments have include improvements in solvent suppression and baseline correction (88) to facilitate both the detection and quantitation of the TRNOE signals and 3D TRNOE measurements (89) to resolve spectral overlap. Other advances include recent reports of heteronuclear (90, 91) and proton X-filtered (92) T R N O E N M R investigations that may further expand the usefulness of the approach. Additional structural information concerning the receptor binding site may be obtained from concentrated protein samples (ca. 1 mM) where weaker protein-ligand intermolecular TRNOEs may be observed (22, 23, 34, 5153, 64, 65, 67). Identification of intermolecular TRNOEs can be facilitated by T i p (56) or T2 (58) filtering to remove protein NOE crosspeaks which are of comparable intensity and linewidth. The Peptidyl Proline Isomerase Cyclophilin. Cyclophilin (CyP) is the 17.8 kDa cytosolic receptor of the immunosuppressive drug cyclosporin A (CsA) and a peptidyl-prolyl cistrans isomerase (PPIase) that catalyzes the cis-trans isomerization of X-Pro imide bonds, a catalysis strongly inhibited by CsA (93-95). While a possible relationship between immunosuppression and PPIase inhibition is unlikely (96, 97), X-Pro isomerization and its catalysis by CyP is, nevertheless, important per se for its role in protein folding (98). Proposed mechanisms for the PPIase catalysis include nucleophilic attack on the carbonyl carbon of the X-Pro peptide bond (99) or protonation of the X-Pro peptide bond nitrogen (100), both leading to the formation of tetrahedral intermediates with decreased double bond



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MOLECULAR MODELING

character for the X-Pro peptide bond and lowered activation energy barrier for the cis-trans interconversion. However, steady-state kinetic investigations of the CyP PPIase activity and specificity (101-103) and sitedirected mutagenesis studies (104) argue against either nucleophilic or acid/base catalysis. According to a third "catalysis by distortion" mechanism (101, 104, 105), non-covalent enzyme-substrate interactions stabilize a substrate transition state with a non-planar X-Pro peptide bond, thereby destroying its resonance stabilization. Thus, the elucidation of the CyP-bound substrate conformation would be particularly revealing with regard to the PPIase mechanism. The x-ray structure of CyP complexed with the model substrate N acetyl-AlaAlaProAla-amidomethylcoumarin (ac-AAPA-amc) at 2.8 Â resolution showed the A P imide bond in the trans conformation with the substrate binding site being identical to that of CsA (706). Subsequent work at 2.3 Â resolution identified the CyP residues in the active site and revealed a structure consisting of a dimer of CyP-substrate complexes, each CyP molecule accommodating a cis ac-AAPA-amc molecule in its active site while also being associated with a partially disordered trans tetrapeptide (107). This dimer arrangement, however, could be the result of the observed stacking of six aromatic groups (four tetrapeptide coumarins and two CyP Trp indoles) and may not be biologically relevant. A third x-ray study at 1.64 Â resolution of CyP complexed with the dipeptide AP, in all likelihood a poor CyP substrate as it is for the PPIase F K B P (103), identified only the cis conformer as protein-bound and provided a detailed description of the protein active site (108). A solvent-assisted catalysis mechanism was suggested that involved the desolvation of the substrate upon entering the hydrophobic CyP active site and its stabilization by a protein-bound water molecule (108). In view of the conflicting reports regarding the CyP-bound substrate conformation, an experimental approach free of artifacts from crystal packing forces was highly desirable and this led to the selection of TRNOE methods for the determination of the CyP-bound substrate conformation in solution. The Selection of a Suitable Substrate. Several Pro-containing oligopeptides are PPIase substrates as evidenced by the appearance of exchange crosspeaks in their NOESY spectra recorded in the presence of PPIase (109). However, not all of them appear to be equally effective, as indicated from competition assays against the standard PPIase substrate (103, 110). In addition to a dependence on the type of amino acid preceding Pro (102), catalysis appears to be affected by the presence of the C-terminal charge and its unfavorable accommodation in the hydrophobic PPIase active site (770). CyP substrates can be quantitatively evaluated by N M R , using saturation transfer methods to measure rate constants (777). The measured CyP-catalyzed cis-trans interconversion rate of ΑΑΡ A was found to be substantially slower than that of the standard CyP substrate N-succinyl-




35

AlaAlaProPhe-paranitroanilide (suc-AAPF-pNA) which may explain the absence of A A P A TRNOEs (Fig. 1). The best estimates of the steady-state kinetic constants for the CyP catalysis of the suc-AAPF-pNA cis-trans isomerism are K M ~ 1 m M and kcat ~ 1.3 χ 10 s" at 0°C {100). The latter value indicates an efficient enzyme and sets a lower limit to all unimolecular steps in the catalytic mechanism, including the dissociation rate of the product from the Michaelis-Menten complex {112), thus satisfying the fast exchange condition for TRNOEs.


4

1

Materials and Methods. Recombinant human CyP was overexpressed in E. coli, harvested, and purified as previously detailed (104). Suc-AAPFpNA was selected as a suitable substrate for the TRNOE studies of the CyP mechanism. Its NMR spectrum was assigned by standard 2D N M R methods (3). Attention was focused on the A2ccH to Ρ3δΗ or P3ocH resonance NOEs which can be used to distinguish between the trans and cis A P conformers (Fig. 2). In the presence of CyP at 25°C, the averaged A2ccH resonance was broadened beyond detection, thus precluding the observation of any NOEs. Experiments were, therefore, conducted at 5°C where the fast exchange condition should still be satisfied. As a result of limited substrate solubility, the more sensitive steady state/truncated driven NOE experiment was used for an assessment of the CyP-bound substrate conformation. NOE difference spectra were acquired with the standard pulse sequence. The residual water resonance was presaturated with a 14 Hz field strength for 2 s and preirradiation at the selected frequency was applied for 400 ms with a field strength of 6 Hz, the strongest one that maintained selectivity of irradiation. Spectra were acquired by subtracting a 16-scan off-resonance control from a 16-scan on-resonance data set and adding the FID differences until a good signal-to-noise ratio in the Fourier transformed difference spectra was achieved after 60 cycles. Each difference spectrum was phased using the parameters of an identically acquired control spectrum phased all positive and referenced to the HDO resonance at 5.02 ppm vs TSP at 5°C. These difference spectra are displayed in Figs. 3-5 and, being identically acquired and processed, can be directly intercompared. The Conformation of the Cyclophilin Substrate. A n important distinction with regard to the conformation of a Pro-containing peptide is whether the X-Pro peptide bond is cis or trans (3). In the trans form, the XccH and ΡδΗ2 protons are in close proximity whereas in the cis form, this is so for the XccH and P a H protons, a difference resulting in distinct, characteristic NOEs for the two conformers (Fig. 2). In aqueous solution, the conformation of the uncomplexed standard CyP model substrate sucA A P F - p N A is ca. 90% trans (100). The cis-trans isomerization is sufficiently slow on the N M R timescale to give rise to two sets of



36

MOLECULAR MODELING

4.0

3.0 F2(ppm)

2.0

4.0

3.0 F2(ppm)

2.0

4.0

3.0

2.0

F2(ppm)

Figure 1. The aliphatic region of the pure phase absorption 500 MHz NOESY spectra (200 ms mixing time) of 10 m M A A P A (A), 1 m M CyP (B), and 10 m M A A P A plus 1 m M CyP (C) in buffered D20 solutions, pH 6.8 at 25°C. The assignments of the dominant trans conformer are provided in A. Exchange crosspeaks due to the CyP-catalyzed, cis-trans interconversion are marked with an arrow in C. However, the substrate exchange between the bound and the free states is not sufficiently fast to produce TRNOEs.

Figure 2. The trans and cis forms of the A-P peptide bond in CH3COAla-Pro-NHCH3. In each form, the broken lines indicate the short distances AocH-P5H2 (trans) and ΑαΗ-ΡαΗ (cis) which give rise to the diagnostic NOEs.




Solution Conformation ofLigands

37

F4 A2

— ι

4.5

1

4.0

1

3.5

1

3.0

1

1

1

2.5

2.0

1.5

f—

1.0

ppm Figure 3. NOE difference spectra (500 MHz) for 0.86 m M suc-AAPFpNA in buffered D2O solutions at pH 6.8 and 5°C. On resonance irradiation frequencies: trans A 2 a H (A), trans Ρ3αΗ (B) and trans Ρ3δΗ (C).



38

MOLECULAR MODELING

1

4.5

1

4.0

1

3.5

1

3.0

1

2.5

1

2.0

1

1.5

1—

1.0

ppm Figure 4. NOE difference spectra (500 MHz) for 0.86 m M suc-AAPFpNA plus 40 μΜ CyP in buffered D2O solutions at p H 6.8 and 5°C. Asterisk-marked peaks (top) are from buffer components (DTT and EDTA). The on-resonance irradiation frequencies are those for the exchange-averaged Α2αΗ (A), P3aH (B), and Ρ3δΗ (C) peaks.



Solution Conformation ofLigands

39


1

J A

1

Β

1

Γ

— ι

4.5

C

Ί

1

1

1

4.0

3.5

2.5

ι

1

1

3.0

2.0

1.5

1—

1.0

ppm Figure 5. NOE difference spectra (500 MHz) for suc-AAPF-pNA (0.86 mM) plus CyP (40 μΜ) plus CsA (80 μΜ) in buffered D2O solutions at pH 6.8 and 5°C. The on-resonance irradiation frequencies are those of the trans A 2 a H (A), trans P3aH (B), and trans Ρ3δΗ (C) peaks.



40

MOLECULAR MODELING

resonances that correspond to a major (trans) and a minor (cis) population in a 10:1 ratio (Fig. 3, top). In the I D N O E difference spectra of free suc-AAPF-pNA, the observed NOEs are small with the exception of that between the geminal Pro5H2 (Fig. 3C) and, surprisingly for a molecule of this size, negative. This may result from slower molecular tumbling at 5°C, particularly in the ca. 40% more viscous D2O solution (Chem. Abstr. 1941, 6169) and perhaps also from molecular association. In the presence of CyP, there is a marked broadening of all peptide resonances (Fig. 4 top) and the cis and trans resonances are averaged as a result of the enhanced CyP-catalyzed cistrans interconversion. No NOE between A2ccH and Ρ3δΗ2 is observed (Figs. 4A and 4C) but there is a significant NOE between A2ocH and P3aH (Figs. 4A and 4B) that must originate from the CyP-bound cis conformer. Upon addition of the PPIase inhibitor CsA, the NOE pattern and intensity (Fig. 5) reverts to that for the unbound substrate (Fig. 3) indicating that the Α2αΗ-Ρ3αΗ TRNOE (Fig. 4) originates from a specific interaction of the substrate with the CyP active site and not from nonspecific substrate binding. The aim of this steady-state NOE study was to determine whether the CyP-bound substrate adopts a cis or a trans conformation. The subsequent quantitative evaluation of additional transient NOE measurements showed that the CyP-bound substrate adopts a cw-like conformation with the A-P peptide bond twisted no more than 40° out of planarity (113). Acknowledgments. This research was supported by the NIH grants GM40660 and GM49858. Literature Cited 1. Blundell, T.L.; Johnson, L.M. Protein Crystallography; Academic Press: New York, 1976. 2. Stezowski, J.J.; Chandrasekhar, K. Annu. Rep. Med. Chem. 1986, 21, 293-302. 3. Wüthrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons, Inc.: New York, 1986. 4. Otting, G. Cur. Opin. Struct. Biol. 1993, 3, 760-768. 5. Fesik, S.W.; Zuiderweg, E.R.P.; Olejniczak, E.T.; Gampe, Jr., R.T. Biochem. Pharmacol. 1990, 40, 161-167. 6. Fesik, S.W. J. Med. Chem. 1991, 34, 2937-2945. 7. Otting, G.; Wüthrich, K. Quart. Rev. Biophys. 1990, 23, 39-96. 8. Hsu, V.L.; Armitage, I.M. Biochemistry 1992, 31, 12778-12784. 9. Neuhaus, D.; Williamson, M.P. The Nuclear Overhauser Effect in Structural and Conformational Analysis; VCH Publishers: New York, 1989. 10. Gronenborn, A.M.; Clore, G.M.; Brunori, M.; Giardina, B.; Falcioni, G.; Perutz, M.F. J. Mol. Biol. 1984, 178, 731-742.





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11. Banerjee, Α.; Levy, H.R.; Levy, G.C.; Chan, W.W.C. Biochemistry 1985, 24, 1593-1598. 12. Leo, G.C.; Castellino, S.; Sammons, R.D.; Sikorski, J.A. Biorg. Med. Chem. Let. 1992, 2, 151-154. 13. Albrand, J.P.; Birdsall, B.; Feeney, J.; Roberts, G.C.K.; Burgen, A.S.V. Int. J. Biol. Macromol. 1979,1,37-41. 14. Feeney, J.; Birdsall, B.; Roberts, G.C.K.; Burgen, A.S.V. Biochemistry 1983, 22, 628-633. 15. Gronenborn, A.M.; Clore, G.M. Biochemistry 1982, 21, 4040-4048. 16. Gronenborn, A.M.; Clore, G.M. J. Mol. Biol. 1982, 157, 155-160. 17. Gronenborn, A.M.; Clore, G.M.; Jeffery, J. J. Mol. Biol. 1984, 172, 559-572. 18. Gronenborn, A.M.; Clore, G.M.; Hobbs, L.; Jeffery, J.; Eur. J. Biochem. 1984, 145, 365-371. 19. Ehrlich, R.S.; Colman, R.F. Biochemistry 1985, 24, 5378-5387. 20. Banerjee, Α.; Levy, H.R.; Levy, G.C.; LiMuti, C.; Goldstein, B.M.; Bell, J.E. Biochemistry 1987, 26, 8443-8450. 21. Andersen, N.H.; Eaton, H.L.; Nguyen, K.T. Magn. Reson. Chem. 1987, 25, 1025-1034. 22. La Mar, G.; Hernandez, G.; de Ropp, J.S. Biochemistry 1992, 31, 9158-9168. 23. Fry, D.C.; Kuby, S.A.; Mildvan, A.S. Biochemistry 1985, 24, 46804694. 24. Rosevear, P.R.; Fox, T.L.; Mildvan, A.S. Biochemistry 1987, 26, 3487-3493. 25. Rosevear, P.R.; Powers, V.M.; Dowhan, D.; Mildvan, A.S.; Kenyon, G.L. Biochemistry 1987, 26, 5338-5344. 26. Landy, S.B.; Ray, B.D.; Plateau, P.; Lipkowitz, K.B.; Rao, B.D.N. Eur. J. Biochem. 1992, 205, 59-69. 27. Williams, J.S.; Rosevear, P.R. J. Biol. Chem. 1991, 266, 2089-2098. 28. Kohda, D.; Kawai, G.; Yokoyama, S.; Kawakami, M.; Mizushima, S.; Miyazawa, T. Biochemistry 1987, 26, 6531-6538. 29. Clore, G.M.; Gronenborn, A.M.; Carlson, G.; Meyer, E.F. J. Mol. Biol. 1986, 190, 259-267. 30. Meyer, E.F., Jr.; Clore, G.M.; Gronenborn, A.M.; Hansen, H.A.S. Biochemistry 1988, 27, 725-730. 31. Balaram, P.; Bothner-By, A.A.; Breslow, E. J. Amer. Chem. Soc. 1972, 94, 4017-4018. 32. Lippens, G.M.; Cerf, C.; Hallenga, K. J. Magn. Reson. 1992, 99, 268281. 33. Nirmala, N.R.; Lippens, G.M.; Hallenga, K. J. Magn. Reson. 1992, 100, 25-42. 34. Lippens, G.; Hallenga, K.; Van Belle, D.; Wodak, S.J.; Nirmala, N.R.; Hill, P.; Russell, K.C.; Smith, D.D.; Hruby, V.J. Biochemistry 1993, 32, 9423-9434.



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35. Ni, F.; Konishi, Y.; Frazier, R.B.; Scheraga, H.A.; Lord, S.T. Biochemistry 1989, 28, 3082-3094. 36. Ni, F., Meinwald, Y.C.; Vásquez, M.; Scheraga, H.A. Biochemistry 1989, 28, 3094-3105. 37. Ni, F.; Konishi, Y.; Bullock, L.D.; Rivetna, M.N.; Scheraga, H.A. Biochemistry 1989, 28, 3106-3119. 38. Ni, F.; Konishi, Y.; Scheraga, H.A. Biochemistry 1990, 29, 44794489. 39. Zheng, Z.; Ashton, R.W.; Ni, F.; Scheraga, H.A. Biochemistry 1992, 31, 4426-4431. 40. Ni, F.; Ripoll, D.R.; Martin, P.D.; Edwards, B.F.P. Biochemistry 1992, 31, 11551-11557. 41. Bushweller, J.H.; Bartlett, P.A. Biochemistry 1991, 30, 8144-8151. 42. Campbell, A.P., Sykes, B.D. J. Mol. Biol. 1991, 222, 405-421. 43. Campbell, A.P.; Van Eyk, J.E.; Hodges, R.S.; Sykes, B.D. Biochim. Biophys. Acta 1992, 1160, 35-54. 44. Landry, S.J.; Gierasch, L.M. Biochemistry 1991, 30, 7359-7362. 45. Landry, S.J.; Jordan, R.; McMacken, R.; Gierasch, L.M. Nature 1992, 355, 455-457. 46. Bevilacqua, V.L; Thomson, D.S.; Prestegard, J.H. Biochemistry 1990, 29, 5529-5537. 47. Bevilacqua, V.L.; Kim, Y.; Prestegard, J.H. Biochemistry 1992, 31, 9339-9349. 48. Andersen, N.H.; Nguyen, K.T.; Eaton, H.L. J. Magn. Reson. 1985, 63, 365-375. 49. Glasel, J.A.; Borer, P.N. Biochem. Biophys. Res. Commun. 1986, 30, 1267-1273. 50. Glasel, J.A. J. Mol. Biol. 1989, 209, 747-761. 51. Anglister, J.; Levy, R.; Scherf, T. Biochemistry 1989, 28, 33603365. 52. Levy, R.; Assulin, O.; Scherf, T.; Levitt, M . ; Anglister, J. Biochemistry 1989, 28, 7168-7175. 53. Anglister,J.;Zilber, B. Biochemistry 1990, 29, 921-928. 54. Zilber, B.; Scherf, T.; Levitt, M.; Anglister, J. Biochemistry 1990, 29, 10032-10041. 55. Scherf, T.; Hiller, R.; Naider, F.; Levitt, M.; Anglister, J. Biochemistry 1992, 31, 6884-6897. 56. Scherf, T.; Anglister, J. Biophys. J. 1993, 64, 754-761. 57. Bruderer, U.; Peyton, D.H.; Barbar, E.; Fellman, J.H.; Rittenberg, M.B. Biochemistry 1992, 31, 584-589. 58. Glaudemans, C.P.J.; Lerner, L.; Daves, Jr., G.D. Kovác, P.; Venable, R.; Bax, A. Biochemistry 1990, 29, 10906-10911. 59. Campbell, A.P.; Tarasow, T.M.; Massefski, W.; Wright, P.E.; Hilvert, D. Proc. Natl. Acad. Sci. USA 1993, 90, 8663-8667. 60. Painter, G.R.; Aulabaugh, A.E.; Andrews, C.W. Biochem. Biophys. Res. Commun. 1993, 191, 1166-1171.



3. KAKALIS & ARMTTAGE


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61. Clore, G.M.; Gronenborn, A.M.; Greipel,J.;Maass, G. J. Mol. Biol. 1986, 187, 119-124. 62. Gronenborn, A.M.; Clore, G.M.; McLaughlin, L.W.; Graeser, E.; Lorber, B.; Giegié, R. Eur. J. Biochem. 1984, 145, 359-364. 63. Ferrin, L.J.; Mildvan, A.S. Biochemistry 1985, 24, 6904-6913. 64. Ferrin, L.J.; Mildvan, A.S. Biochemistry 1986, 25, 5131-5145. 65. Plensniak, L.A.; Boegeman, S.C.; Segelke, B.W.; Dennis, E.A. Biochemistry 1993, 32, 5009-5016. 66. Behling, R.W.; Yamane, T.; Navon, G.; Jelinski, L.W. Proc. Natl. Acad. Sci. USA 1988, 85, 6721-6725. 67. Fraenkel, Y.; Gershoni, J.M.; Navon, G. FEBS Let. 1991, 291, 225228. 68. Dratz, E.A.; Furstenau, J.E.; Lambert, C.G.; Thireault, D.L.; Rarick, H.; Schepers, T.; Pakhlevaniants, S.; Hamm, H.E. Nature 1993, 363, 276-281. 69. Sukumar, M.; Higashijima, T. J. Biol. Chem. 1992, 267, 2142121424. 70. Wakamatsu, K.; Higashijima, T.; Fujino, M.; Nakajima, T.; Miyazawa, T. FEBS Let. 1983, 162, 123-126. 71. Wakamatsu, K; Okada, Α.; Higashijima, T.; Miyazawa, T. Biopolymers 1986, 25, S193-S200. 72. Wakamatsu, K.; Okada, Α.; Miyazawa, T.; Ohya, M.; Higashijima, T. Biochemistry 1992, 31, 5654-5660. 73. Wakamatsu, K.; Okada, Α.; Miyazawa, T.; Masui, Y.; Sakakibara, S.; Higashijima, T. Eur. J. Biochem. 1987, 163, 331-338. 74. Kuroda, Y.; Kitamura, K. J. Amer. Chem. Soc. 1984, 106, 1-6. 75. Milon, Α.; Miyazawa, T.; Higashijima, T. Biochemistry 1990, 29, 65-75. 76. Clore, G.M.; Gronenborn, A.M. J. Magn. Reson. 1982, 48, 402-417. 77. Clore, G.M.; Gronenborn, A.M. J. Magn. Reson. 1983, 53, 423-442. 78. Rosevear, P.R.; Mildvan, A.S. Methods Enzymol. 1989, 177, 333358. 79. Campbell, A.P.; Sykes, B.D. Annu. Rev. Biophys. Biomol. Struct. 1993, 22, 99-122. 80. Campbell, A.P.; Sykes, B.D. J. Magn. Reson. 1991, 93, 77-92. 81. Campbell, A.P.; Sykes, B.D. J. Biomol. NMR 1991, 1, 391-402. 82. London, R.E.; Perlman, R.E.; Davis, D.G. J. Magn. Reson. 1992, 97, 79-98. 83. Landy, S.B.; Rao, B.D.N. J. Magn. Reson. 1989, 81, 371-377. 84. Ni, F. J. Magn. Reson. 1992, 96, 651-656. 85. Akasaka, K. J. Magn. Reson. 1979, 36, 135-140. 86. Dubois, B.W.; Evers, A.S. Biochemistry 1992, 31, 7069-7076. 87. Zheng,J.;Post, C.B. J. Magn. Reson. Β 1993, 101, 262-270. 88. Ni, F. J. Magn. Reson. 1992, 99, 391-397. 89. Ni, F. J. Magn. Reson. 1992, 100, 391-400.


MOLECULAR MODELING

44


90.

Batta, G.; Kövér, K.E.; Székely, Ζ.; Sztaricskai, F. J. Amer. Chem. Soc. 1992, 114, 2757-2758. 91. Wang, S.X.; Caines, G.H.; Schleich, T. J. Magn. Reson. B 1993, 102, 47-53. 92. Oschkinat, H.; Schott, K.; Bacher, A. J. Biomol. NMR 1992, 2, 1932. 93. Handschumacher, R.E.; Harding, M.W.; Rice, J.; Drugge, R.J. Science 1984, 226, 544-547. 94. Takahashi, N.; Hayano, T.; Suzuki, M. Nature 1989, 337, 473-475. 95. Fischer, G.; Wittmann-Liebold, B.; Lang, K.; Kiefhaber, T.; Schmid, F.X. Nature 1989, 337, 476-478. 96. Walsh, C.T.; Zydowsky, L.D.; McKeon, F.D. J. Biol. Chem. 1992, 267, 13115-13118. 97. Zydowsky, L.D.; Etzkorn, F.A.; Chang, H.Y., Ferguson, S.B.; Stolz, L.A.; Ho, S.I.; Walsh, C.T. Protein Sci. 1992, 1, 1092-1099. 98. Schmid, F.X., Mayr, L.M.; Mücke, M.; Schönbrunner, E.R. Adv. Protein Chem. 1993, 44, 25-66. 99. Fischer, G.; Berger, E.; Bang, H. FEBS Let. 1989, 250, 267-270. 100. Kofron, J.L.; Kuzmic, P.; Kishore, V.; Colón-Bonilla, E.; Rich, D.H. Biochemistry 1991, 30, 6127-6134. 101. Harrison, R.K.; Stein, R.L. Biochemistry 1990, 29, 1684-1689. 102. Harrison, R.K.; Stein, R.L. Biochemistry 1990, 29, 3813-3816. 103. Harrison, R.K.; Stein, R.L. J. Amer. Chem. Soc. 1992, 114, 34643471. 104. Liu,J.;Albers, M.W.; Chen, C.-M.; Schreiber, S.L.; Walsh, C.T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2304-2308. 105. Stein, R.L. Adv. Protein Chem. 1993, 44, 1-24. 106. Kallen, J.; Spitzfaden, C.; Zurini, M.G.M.; Wider, G.; Widmer, H.; Wüthrich, K.; Walkinshaw, M.D. Nature 1991, 353, 276-279. 107. Kallen,J.;Walkinshaw, M.D. FEBS Let. 1992, 300, 286-290. 108. Ke, M.; Mayrose, D.; Cao, W. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3324-3328. 109. Justice, Jr., R.M.; Kline, A.D.; Sluka, J.P.; Roeder, W.D.; Rodgers, G.H.; Roehm, N.; Mynderse, J.S. Biochem. Biophys. Res. Commun. 1990, 171, 445-450. 110. Park, S.T.; Aldape, R.A.; Futer, O.; DeCenzo, M.T.; Livingston, D.J. J. Biol. Chem. 1992, 267, 3316-3324. 111. Hsu, V.L.; Handschumacher, R.E.; Armitage, I.M. J. Amer. Chem. Soc. 1990, 112, 6745-6747. 112. Hammes, G.G. Enzyme Catalysis and Regulation; Academic Press: New York, 1982; pp. 38-42. 113. Kakalis, L.T.; Armitage, I.M. Biochemistry 1993, in press. RECEIVED January 13, 1994


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