Proc. Natl. Acad. Sci. USA Vol. 75, No. 11, pp. 5281-5285, November 1978 Biochemistry A photo-CIDNP study of the interaction of oligonucleotides with gene-5 of bacteriophage M13 (nuclear magnetic resonance/chemically induced dynamic nuclear polarization/DNA-protein interactions/) G. J. GARSSENt f, R. KAPTEIN§, J. G. G. SCHOENMAKERSt, AND C. W. HILBERSt t Department of Biophysical Chemistry, I Department of Molecular Biology, University of Nijmegen, Nijmegen, The Netherlands; and § Department of Physical Chemistry, State University, Gronigen, The Netherlands Communicated by R. G. Shulman, July 31, 1978

ABSTRACT It is shown that photo-CIDNP effects (CIDNP, Here 'F and 3F represent the flavin in the excited singlet and chemically induced dynamic nuclear polarization) can be triplet states, respectively. CIDNP effects (indicated by an as- generated in the 360-MHz proton NMR spectrum of gene-5 protein from bacteriophage M13. This technique is used to de- terisk) arise from the spin-selective recombination of the radical termine the number of tyrosyl residues at the surface of the pair (reaction 3). For N-acetyltyrosine a strong emission effect protein and to assign the resonances from the 3,5-ring protons has been observed for the 3,5-ring protons (ortho with respect of these residues. The DNA-binding site of the protein is in- to the hydroxyl group) and weak enhanced absorption for the vestigated by formation of complexes with oligonucleotides. 2,6-ring protons and the f3-CH2 protons (10). The method Complex formation leads to shifting and/or quenching of the discriminates between photo-CIDNP emission signals of the surface , implying exposed and buried amino acid side that they are involved in DNA-protein interaction. These ex- chains. This has been demonstrated in the case of bovine pan- periments are complemented by studying the complex forma- creatic trypsin inhibitor (11). tion of Lys-Tyr-Lys to poly(A). EXPERIMENTAL Gene-5 protein (G-VP) encoded by the filamentous coliphage M13 has been shown to bind strongly and cooperatively to Materials and Methods. The isolation and purification of single-stranded DNA (1, 2). As a consequence, it is able to de- G-VP were as previously described (9). G-VP containing deu- stabilize double-stranded DNA material (3). These properties terated phenylalanine ([2H7]Phe-substituted G-VP) was pre- of the protein underlie its regulating function during synthesis pared by infecting E. coli with phage in the presence of deu- of single-stranded viral DNA from the double-helical circular terated phenylalanine while the aromatic amino acid synthetic replicative form in the infected Escherichia coil cell. The pathway in E. coli was suppressed (details will be published stoichiometry of the G-VP-DNA complex in vitro is such that elsewhere). d(pC-G-C-G) was purchased from Collaborative one protein monomer covers four nucleotides in single-stranded Research (Waltham, MA) and converted to the Na+ form (9). DNA (3-5). The protein has a molecular weight of 9690. It N-Acetyltyrosine and r(A-A) (NH4+ salt, E = 2.7 X 104 M-1 occurs as a dimer under a wide range of conditions, the binding cm-1 at 260 nm, pH = 7.0) were obtained from Boehringer forces being mainly hydrophobic (5, 6). The amino acid se- (Mannheim, West Germany) as was poly(A) (K+ salt), which quence of the protein has been determined (7, 8). For the dis- contained 2.3 ,umol of P per mg. L-Lysyl-L-tyrosyl-L-lysine cussion in this paper it is relevant to know that G-VP contains (diacetate, hemihydrate) was from Schwarz/Mann. Its con- one histidyl, three phenylalanyl, and five tyrosyl residues. centration was determined spectrophotometrically in 0.1 M In a recent NMR study we investigated kinetic aspects of the NaOH (12). protein-induced "unwinding" of the self-complementary 3-N-Carboxymethyllumiflavin used in the laser photo- double-stranded d(pC-G-C-G) (9). In the present paper we start CIDNP experiments was a gift from F. Muller (Wageningen, to investigate the nature of the interactions between the protein The Netherlands). The tripeptide and the nucleotides were used and the DNA. These investigations are complemented by ex- without further purification. All other materials were of reagent periments on the Lys-Tyr-Lys-poly(A) complex. To this end we grade. have employed the recently developed laser photo-CIDNP Instrumentation. Absorption spectra were recorded on a method (CIDNP, chemically induced dynamic nuclear po- Zeiss PMQII spectrophotometer. The notation pH* denotes larization) (10, 11). Essentially in this method NMR lines be- uncorrected pH meter reading in 2H20 solutions. longing to certain amino acid side chains (tyrosine, , 360-MHz NMR spectra were taken on a Bruker HX-360 and ) are selectively enhanced when these residues spectrometer operating in the pulse Fourier transform mode. are situated at the surface of the protein. Nuclear spin polar- Chemical shifts, 6, are quoted relative to the internal standard ization is generated in a reversible reaction with a flavin dye, sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). All which is photo-excited in the NMR probe by irradiation with spectra were taken from solutions in 2H20 and consist of 25 an argon laser. In the case of tyrosyl residues (TyrH) the phe- scans unless indicated otherwise. The difference method for nolic hydrogen atom is abstracted by the triplet flavin. The the detection of photo-CIDNP in biological macromolecules reaction can be written: has been described in detail elsewhere (10). Briefly, the sample hv F IF--3F [1] is irradiated in the probe of the HX-360 NMR spectrometer with light pulses from a Spectra Physics model 171 argon ion 3F + TyrH o FH + Tyr- [2] laser (multiline, 7-W). Alternating "light" and "dark" free induction decays are collected, which can be subtracted to yield FH + Tyr o F + TyrH* [3] the pure CIDNP spectrum. The light spectrum is taken im-

The publication costs of this article were defrayed in part by page Abbreviations: CIDNP, chemically induced dynamic nuclear polar- charge payment. This article must therefore be hereby marked "ad- ization; G-VP, gene-5 protein. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate t Present address: Research Institute for Animal Husbandry, "Schoo- this fact. noord," Driebergseweg 10d, Zeist, The Netherlands. 5281 Downloaded by guest on September 25, 2021 5282 Biochemistry: Garssen et al. Proc. Natl. Acad. Sci. USA 75 (1978) mediately after the laser pulse. Before recording of both the these conditions only one strong emission signal is found at a light and dark free induction decays, a broadband presaturation position of 6.8 ppm, where the 3,5-ring protons of free tyrosine radio-frequency pulse is given. For both spectra the pulse se- resonate. The small emission lines at 2.6 and 4.2 ppm belong quence is the same except that for the dark spectrum the light to the flavin 8- and 10-methyl groups, respectively. The en- pulse is replaced by an equally long delay. Typical operating hanced absorption around 3 ppm in spectra C and D is due to conditions are: 0.4-s laser pulse, 1-s presaturation pulse, 7-,us tyrosine f3-CH2 protons. radio-frequency pulse corresponding with a 900 flip angle, 1-s The CIDNP effects in G-VP are markedly dependent on the acquisition time (4000-Hz spectral window), 15-s repetition protein concentration. This is demonstrated in Fig. 2, in which time for one cycle (time between laser pulses). the aromatic parts of the CIDNP difference spectra are pre- sented for protein concentrations ranging from 0.09 to 1.54 mM. RESULTS At a concentration of 1.54 mM G-VP three resonances at 6.49, Assignments. Fig. 1 shows the effects of a photo-CIDNP 6.75, and 6.92 ppm of about the same intensity are observed in experiment in the -360-MHz proton NMR spectrum of the emission; moreover, a shoulder is present at 7.03 ppm. At de- [2H7]Phe-substituted G-VP. The CIDNP effect is represented creasing protein concentrations the peak at 6.92 ppm shifts in spectrum C of Fig. 1 as the difference between the spectra upfield and coincides with the resonance that started at 6.75 A (light spectrum) and B (dark spectrum). Exactly the same ppm. In addition the weak emission at 7.03 ppm shifts down- CIDNP difference spectrum was obtained with G-VP con- field over 0.1 ppm. Interestingly, the intensity of the emission taining protonated phenylalanines (data not shown). It is clear signals increases at decreasing protein concentrations. In that the emission signals at 6.52 and 6.84 ppm are the most spectrum C of Fig. 2 the intensity ratio of the two major emis- outstanding features in this difference spectrum. The signal at sion signals becomes 2:1. At the lowest concentration the 4.9 ppm arises from a slight shift of the 1H2HO resonance due amount of protein available becomes so low as to be the limiting to heating of the sample by the laser pulse. Spectrum D of Fig. factor to the signal intensity. Because the aromatic region of the 1 shows the CIDNP difference spectrum of the protein dena- spectrum consists of the ring proton resonance of only one tured in a solution containing 5.6 M guanidine-2HCI. Under histidine and five tyrosines, the strong emission lines in Figs. 1 and 2 must belong to tyrosine 3,5-ring protons, in agreement with the finding in bovine pancreatic trypsin inhibitor (11). From spectrum A of Fig. 2 we further conclude that there are

A

C

D I

ppm FIG. 1. 360-MHz proton NMR spectra of [2H7]Phe-substituted G-VP. Spectrum A was recorded after the light pulse; spectrum B was obtained without light pulse; C is the difference spectrum A - B. 9 8 7 6 5 Conditions: 0.75 mM protein, 0.87 mM Tris-2HCI, 43 mM NaCI, 0.24 ppm mM flavin, pH* 7.3, ambient temperature. D is the CIDNP difference FIG. 2. Aromatic part of the photo-CIDNP difference spectrum spectrum of G-VP in the presence ofguanidine-2HCl. Conditions: 0.41 of G-VP recorded at 360 MHz at different protein concentrations. A, mM protein, 0.23 mM Tris, 11 mM NaCl, 0.23 mM flavin, 5.6 M 1.54 mM; B, 0.75 mM; C, 0.38 mM; D, 0.09 mM. Other conditions as guanidine-2HCI, ambient temperature. for spectra A-C of Fig. 1. Downloaded by guest on September 25, 2021 Biochemistry: Garssen et al. Proc. Natl. Acad. Sci. USA 75 (1978) 5283 three tyrosines that are CIDNP active. They are situated at the not show any CIDNP effect. To ensure that the total amount surface of the protein and apparently have about equal acces- of protein was complexed to the DNA fragment, an excess of sibility to the photo-excited dye (Fig. 2, spectrum C). The tetranucleotide was present in solution [0.38 mM protein versus shoulder observed at 7.07 ppm in spectrum C of Fig. 1 and at 0.46 mM d(pC-G-C-G)]. The possibility that the flavin is in- 7.1 ppm in spectrum C of Fig. 2 probably belongs to tyrosine activated in the presence of the oligonucleotide was excluded 2,6-ring protons polarized by polarization transfer from the by adding a small amount of N-acetyltyrosine (0.28 mM) to the 3,5-ring protons (see Discussion). solution. Under these conditions the emission signal of a free C-VP Complexed to Nucleic Acid. From circular dichroism tyrosine appears in the CIDNP difference spectrum (Fig. 3, and quenching studies (4, 5) it has been inferred spectrum D). This observation renders unlikely the possible that tyrosyl residues participate in the protein-nucleic acid unavailability of the flavin in the experiment of Fig. 3, spectrum binding process. Therefore, it is interesting to see how the C. This conclusion is corroborated by the experiment presented CIDNP signals of the three tyrosines at the surface of the pro- in Fig. 4, in which the binding of r(A-A) to the protein is studied. tein are influenced by complex formation with DNA. We Because the protein covers four nucleotides, smaller fragments choose the self-complementary tetranucleotide d(pC-G-C-G) are not expected to shield equally all three tyrosines at the as a substrate for two reasons. First, it is known that the protein protein surface, provided such fragments bind preferentially covers four nucleotides when bound to DNA (3-5). Second, to a specific part of the total DNA binding site. At increasing much of the binding and unwinding influence of the protein r(A-A) concentrations the tyrosine emission, originally at 6.52 with respect to this tetranucleotide is already known (9). ppm, shifts upfield (0.2 ppm) and its intensity decreases, while Spectra A and B of Fig. 3 are the aromatic region of the the emission signal at 6.8 ppm is virtually unaffected. normal and the photo-CIDNP difference spectrum of [2H7]- Complex of Lys-Tyr-Lys with Poly(A). The measurements Phe-substituted G-VP in the absence of d(pC-G-C-G). The on G-VP oligonucleotide complexes were complemented by CIDNP difference spectrum of the G-VP-tetranucleotide studies of the tripeptide Lys-Tyr-Lys in combination with complex is shown in spectrum C of Fig. 3. It is clear that the poly(A). In Fig. 5, spectrum A, the NMR spectrum (dark tyrosine emission signals have disappeared from the spectrum. spectrum) of Lys-Tyr-Lys is shown. We shall not give a com- In a separate experiment it was found that d(pC-G-C-G) did plete interpretation of the spectrum but merely note that the tyrosine 3,5-ring protons resonate at 6.9 ppm, the 2,6-ring protons at 7.22 ppm, and the f3-CH2 protons around 3 ppm. A Because of the saturation pulse given initially (see Materials

A

B

C

D

W-nw-1

104 9 8 7 6 ppm FIG. 3. Effect ofthe binding ofthe tetranucleotide d(pC-G-C-G) on the aromatic part of the 360-MHz proton spectrum of G-VP. A is the spectrum of the [2H7]Phe-substituted G-VP (400 accumulations); B is the CIDNP difference spectrum; solvent conditions as for spectra 8 7 6 5 A-C of Fig. 1. C is the CIDNP difference spectrum of the G-VP ppm complexed to d(pC-G-C-G); conditions: 0.38 mM protein, 0.46 mM FIG. 4. Effect ofr(A-A) binding upon the photo-CIDNP signals d(pC-G-C-G), 0.62 mM Tris, 32 mM NaCl, 0.24 mM flavin, 1 mM of the aromatic part of the 360-MHz proton spectrum of G-VP. EDTA, pH* 6.9, ambient temperature. D is as C, but with 0.28 mM Conditions: 0.37 mM protein, 1.3 mM Tris, 0.24 mM flavin, 34 mM N-acetyltyrosine added. NaCl. Ratio r(A-A)/G-VP: A, 0.0; B, 4.6; C, 11.4; D, 17.3. Downloaded by guest on September 25, 2021 5284 Biochemistry: Garssen et al. Proc. Natl. Acad. Sci. USA 75 (1978) spectrum of the Lys-Tyr-Lys-poly(A) complex. The tyrosyl 3,5-ring protons are in emission and remain shifted upfield, while compared to spectrum B the emission intensity is reduced to 30%. The tyrosyl fl-CH2 protons around 3.0 ppm show en- hanced absorption and have also been shifted upfield. Inter- A estingly, in spectrum D of Fig. 5 the tyrosine 2,6-ring protons (shifted from 7.22 to 7.05 ppm) do not show enhanced ab- sorption as in spectrum B, but instead show a smaller emission B effect. We shall return to this later. DISCUSSION In the present paper the CIDNP method has been employed to detect the resonances of 3,5-ring protons of tyrosines at the surface of G-VP and to probe its DNA binding site. The [2H7]Phe-substituted G-VP provides a particularly simple system, because the aromatic part of its NMR spectrum contains only resonances of a single histidine and five tyrosines; the C protein does not contain tryptophan. From the experiments in Fig. 1 it is evident that only tyrosyl residues give rise to signif- icantly polarized signals. The single histidine is not affected by the photoreaction. The experiments in Fig. 2 demonstrate that only three out of the total of five tyrosyl residues take part in the photo-CIDNP reaction. These results are in excellent agreement with the experiments of Anderson et al. (14), who found that, in the native G-VP, three out of the five tyrosine residues can be modified chemically by tetranitromethane and by N-acetylimidazole. It was shown that these tyrosines cor- respond with the numbers 26, 41, and 56 in the primary se- D quence of the protein (14). We therefore assign the resonance at 6.52 and part of the resonances at 6.8 ppm to the 3,5-ring protons of these tyrosines. Comparison of the spectrum of the [2H7]Phe-substituted G-VP (Fig. 3) with the spectrum of G-VP containing protonated phenylalanines (9, 15) shows that the resonances around 7.4 ppm have to be assigned to the phenyl- alanine ring protons. These results are partly at variance with assignments proposed by Coleman et al. (15), who attributed the resonances around 7.4 ppm to tyrosine ring protons, on the basis of shifts of these resonances induced upon binding of DNA 9 8 7 6 5 4 3 2 1 fragments. Thus in addition to shielding the three tyrosyl ppm residues at the protein surface, DNA binding also affects the phenylalanine residues. The concentration dependence of the FIG. 5. Photo-CIDNP effects in the 360-MHz proton NMR CIDNP effect and of the shifts of the tyrosine protons observed spectrum of Lys-Tyr-Lys and of its complex with poly(A). A, Dark in Fig. 2 are indicative of association of the G-VP dimers, which spectrum Lys-Tyr-Lys; B, the tripeptide's photo-CIDNP difference spectrum; C, dark spectrum poly(A); D, dark spectrum of mixture of have the tendency to form higher aggregates (5, 6). Normally poly(A) and Lys-Tyr-Lys; E, corresponding CIDNP difference one would expect a decrease of the CIDNP when the concen- spectrum. Conditions: 8.7 mM Lys-Tyr-Lys, 24 mM poly(A), 8.6 mM tration is lowered, because the effect arises from a second-order Na cacodylate, 0.9 mM NaCi, 1.7 mM EDTA, 0.24 mM flavin, ambient reaction with the triplet flavin. The observed increase of the temperature. All spectra consist of 13 accumulations. The vertical CIDNP intensities therefore reflects a partial deblocking of the scale of spectra D and E is different from that of spectra A, B, and tyrosines, probably by a shift in the tetramer-dimer equilib- C. rium. By using the CIDNP effect the DNA-binding site of the and Methods) not all of the peak intensities have reached their protein can be investigated. The experiments presented in Fig. normal values. B of Fig. 5 is the CIDNP difference spectrum 3 show that upon binding of the tetranucleotide d(pC-G-C-G) of the tripeptide. Obviously the tyrosyl 3,5-ring protons are in the tyrosine emission signals are completely quenched, dem- strong emission, while the 2,6-ring protons and the f3-CH2 of the three are protons show enhanced absorption. The proton NMR spectrum onstrating that the hydroxyl groups tyrosines C. of the no longer available for reaction with the flavin, either because of poly(A) is given in Fig. 5, spectrum Assignments or because of some other effect. as H8 at 7.82 ppm and H2 at of hydrogen bonding shielding resonances are follows: adenine and 56 are Hi' at 5.54 ppm The at 4.56 ppm This we interpret to mean that the tyrosines 26, 41, 7.74 ppm, ribose (13). peaks interaction. These results are are from other sugar protons but have not been assigned. In Fig. involved in the protein-DNA Anderson et al. 5, D is the dark spectrum of a mixture of 8.7 mM Lys-Tyr-Lys again in excellent agreement with those of (14), and 24 mM poly(A). Comparison with spectra A and B shows who showed that in the in vitro complex of G-VP with single- that the resonance of the tyrosyl ring protons shifts upfield (0.17 stranded DNA the reaction of the three tyrosyl residues with ppm), while downfield shifts are observed for the adenine H8 tetranitromethane is completely abolished. Because the protein resonance (0.18 ppm) and the adenine H2 resonance (0.06 covers approximately four nucleotides in the in vitro protein- ppm). In addition, the ribose H1' proton resonance shifts DNA complex and because the tetranucleotide d(pC-G-C-G) downfield (0.16 ppm). In Fig. 5, E is the CIDNP difference shields the three tyrosines at the surface of the protein, it is Downloaded by guest on September 25, 2021 Biochemistry: Garssen et at. Proc. Natl. Acad. Sci. USA 75 (1978) 5285 expected that not all of these tyrosines in the DNA-binding sites tion has the same sign as that observed for large molecules. The can be covered by a dinucleotide. Indeed, this follows from the present experiments illustrate various possibilities of how. experiments presented in Fig. 4, showing the effect of the photo-CIDNP experiments can be employed in studying pro- binding of r(A-A). Because of the low association constant a teins and protein-nucleic acid interactions in solution. As- considerable excess of r(A-A) had to be added in order to ob- signment of resonances of amino acid residues in can serve its shielding effect. Surprisingly, only the emission signal be made and their accessibility tested. Presently this is restricted at 6.52 ppm is significantly affected. This means that the to resonances of the aromatic amino acids tyrosine, tryptophan, binding takes place preferentially at one of the tyrosines (res- and histidine, but possibly the method can be extended to other onance at 6.52 ppm) and not randomly over the DNA-binding amino acids. site. This suggests to us that it is just this part of the protein molecule that first hooks onto DNA in the course of the asso- ciation process. Additional information is We are indebted to Dr. R. N. H. Konings for the provision of tech- provided by the ex- nical facilities and the gift of the bacteriophage. We thank Dr. F. periments on the complexation of Lys-Tyr-Lys with poly(A). Muller for the lumiflavin. K. Dijkstra is acknowledged for his excellent This system has been used as a model for protein-nucleic acid technical assistance. This work was supported by the Netherlands interaction by H6lene and his coworkers (16-18), who showed Foundation for Chemical Research (SON) with financial aid from the that complexation results in upfield shifts of the tyrosine ring Netherlands Organization for the Advancement of Pure Research proton resonances and downfield shifts of the adenine H8 (ZWO). We wish to acknowledge ZWO for support of the 360-MHz proton resonances as a result of stacking of the tyrosine rings NMR facility at Groningen. onto the adenines in poly(A). Analogous observations have been made in the present experiments (see Fig. 5). In addition, the shifts of the tyrosine resonances observed for the complex (Fig. 1. Denhardt, D. T. (1975) Crit. Rev. Microbiol. 4,161-223. 5, spectrum D) are retained in the emission signal of the CIDNP 2. Ray, D. S. (1977) in Comprehensive Virology, eds. Frankel- experiment (Fig. 5, spectrum E). Moreover the intensity of the Conrat, H. & Wagner, R. (Plenum, New York), Vol. 7, pp. emission signal is reduced to 30% of that observed for the un- 105-178. 3. Alberts, B., Frey, L. & Delius, H. (1972) J. Mol. Biol. 68, 139- complexed tripeptide (Fig. 5, spectrum B). This can be ex- 152. plained by noting that the tripeptide is in fast exchange between 4. Day, L. A. (1973) Biochemistry 12,5329-5339. free and bound states. If the tyrosyl residue is polarizable only 5. Pretorius, H. T., Klein, M. & Day, L. A. (1975) J. Biol. Chem. 250, when the peptide is free in solution and not when bound to the 9262-9269. polynucleotide, it can account for the observed reduction in 6. Cavalieri, S. J., Neet, K. E. & Goldthwait, D. A. (1976) J. Mol. CIDNP intensity. An analogous effect is observed for the Biol. 102, 697-711. complex formation of r(A-A) to G-VP. The tyrosine emission 7. Cuyper, T., Van der Ouderaa, F. J. & De Jong, W. W. (1974) signal at 6.52 ppm shifts and diminishes in intensity with in- Biochem. Biophys. Res. Commun. 59,557-564. creasing concentration of 8. Nakashima, Y., Dunker, A. K., Marvin, D. A. & Konigsberg, W. r(A-A). When the binding becomes (1974) FEBS Lett. 43, 125. strong and the binding equilibrium is shifted towards the nu- 9. Garssen, G. J., Hilbers, C. W., Schoenmakers, J. G. G. & Van cleic acid-protein complex as in the case of d(pC-G-C-G) (see Boom, J. H. (1977) Eur. J. Biochem. 81, 453-463. Fig. 3), access to the tyrosines is completely blocked and no 10. Kaptein, R., Dijkstra, K., Muiller, F., Van Schagen, C. G., & Visser, polarization is observed. A. J. W. G. (1978) J. Magn. Reson. 31, 171-176. CIDNP effects can be influenced by cross-relaxation effects. 11. Kaptein, R., Dijkstra, K. & Nicolay, K. (1978) Nature (London) This is found for the tyrosine 2,6-ring protons. For small mol- 274,293-294. ecules in the fast tumbling limit (see Fig. 5, spectrum B) these 12. Beaven, G. H. & Holiday, E. R. (1952) Advances in Protein protons give rise to enhanced absorption. Polarization transfer Chemistry, eds. Anfinsen, C. B., Jr., Anson, M. I., Bailey, K. & Edsall, J. T. (Academic, New York), Vol. 7, 319-386. causes polarization opposite in sign to that of the primary po- 13. Bovey, F. A. (1972) High Resolution NMR ofMacromolecules larization (at the 3,5-tyrosine ring protons). For large molecules (Academic, New York). with long correlation times the transferred polarization has the 14. Anderson, R. A., Nakashima, Y. & Coleman, J. E. (1975) Bio- same sign, giving rise to enhanced emission as demonstrated chemistry 14, 907-917. in the CIDNP spectra of G-VP and of bovine pancreatic trypsin 15. Coleman, J. E., Anderson, R. A., Ratcliffe, R. G. & Armitage, I. inhibitor (11). In the case of the binding of Lys-Tyr-Lys to M. (1976) Biochemistry 15,5419-5430. poly(A) the cross-relaxation rate is an average of the rates of the 16. Helene, C. & Dimicoli, J. J. (1972) FEBS Lett. 26,6-10. free and bound peptide. Apparently under the conditions of 17. Brun, F., Toulme, J. J. & Helene, C. (1975) Biochemistry 14, the 558-563. experiment of Fig. 5, spectrum E, the average cross-relax- 18. Durand, M., Maurizot, J. C., Borazan, H. N. & H6lene, C. (1975) ation rate has changed sign such that the transferred polariza- Biochemistry 14, 563-570. Downloaded by guest on September 25, 2021