Conformational dynamics in fluorophenylcarbamoyl-alpha-chymotrypsins

A series of fluorine-substituted diphenylcarbamoyl chlorides have been synthesized and used to prepare corresponding diphenylcarbamoylated derivatives of alpha-chymotrypsin. The enzyme is rapidly inactivated by these compounds, as has been previously observed for the unsubstituted chloride, and the derivatives are stable enough to permit extensive studies by fluorine NMR spectroscopy. In combination with previously reported results, these NMR experiments suggest that the aromatic rings of a diphenylcarbamoyl group attached to chymotrypsin may be found in two magnetically and dynamically distinguishable sites, with exchange between these sites taking place by a process that involves rotation about the carbamoyl N-CO bond and localized unfolding of the enzyme. The extent to which a given fluoroaromatic ring is found in one of these sites is dependent on the position of the fluorine substituent and the nature of the partner aromatic ring. It is found that a 2-fluorophenyl ring, when present, dominantly determines site occupation, while a 3-fluorophenyl ring has no effects that are detectably different from those of an unsubstituted phenyl ring. There is evidence for slow aromatic ring rotation within at least one of the phenyl ring interaction sites. Saturation transfer and lineshape methods provide information about the rates of interconversion of the N-phenyl groups between these sites. Line-width, spin-lattice relaxation times and fluorine-proton nuclear Overhauser effects determined at 282 and 470 MHz are reported for each system examined.     

Complete tyrosine assignments in the high field 1H nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor.

The low-field portions of the 250-MHz 1H nuclear magnetic resonance (NMR) specra of native and chemically modified bovine basic pancreatic trypsin inhibitor (BPTI) have been studied as a function of pH over the range pH 5-13. Resonances associated with the 16 protons of the aromatic rings of the four BPTI tyrosines have been located and assigned to specific tyrosyl residues. Titrations of pH yielded pK's for tyrosines-10, -21, -23, and -35 of 10.4, 11.0, 11.7, and 11.1, respectively. The resonances associated with the nitrotyrosine-10 protons of mononitrated BPTI and the nitrotyrosine-10 and -21 protons of dinitrated BPTI have been similarly located, assigned and titrated yielding pK's for nitrotyrosine-10 and -21 of 6.6 and 6.4, respectively. The high-field NMR spectrum indicates that the aromatic ring of tyrosine-35 rotates less than 160 times per second at 25 degrees for pH's in the range 5-9.     

Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). II. Semi-empirical energy calculations.

The molecular conformation of the basic pancreatic trypsin inhibitor (BPTI) is known in considerable detail from both X-ray studies in single crystals and NMR studies in solution. The NMR experiments showed that the aromatic rings of the phenylalanyl and tyrosyl residues can undergo rapid rotational motions about the C beta--Cv bond. The present paper describes a model investigation of the mechanistic aspects of these intramolecular rotational motions. From calculations of the conformational energies for molecular species derived from the X-ray structure by rotations of individual aromatic rings, it was apparent that the rotational motions of the aromatics could only be understood in a flexible structure. Flexibility was simulated by allowing the protein to relax to an energetically favorable conformation for each of the different rotation states of the aromatic rings. It was then of particular interest to investigate how the perturbations caused by different rotation states of the aromatic rings were propagated in the protein structure. It was found that the rotation axes C beta--Cv were only slightly affected (delta X1 approximately less than 20 degrees. The most sizeable perturbations are caused by through space interactions with nearby atoms, which move away from the ring center and thus release the steric hindrance opposing the rotational motions. The values for the energy barriers obtained from the energy minimization are of the same order of magnitude as those measured by NMR.     

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). I. 1H NMR studies.

The basic pancreatic trypsin inhibitor (BPTI) was investigated by high resolution 1H NMR techniques at 360 MHz. Observation of the amide proton resonances of the polypeptide backbone showed that the globular conformation of BPTI determined by X-ray studies in single crystals is maintained in aqueous solution over the temperature range from 4 degrees to 87 degrees. NMR studies over this temperature range of the aromatic amino acid residues of BPTI. i.e. 4 tyrosines and 4 phenylalanines, led to complete assignments of all the aromatic spin systems in the protein. From this, information was obtained on the rotational motions about the C beta--Cv bond axis of the aromatic rings in the globular form of PBTI. At 25 degrees, two tyrosine rings and one phenylalanine ring are rotating rapidly on the NMR time scale. For the other rings the transitions from slow to rapid rotational motions were investigated at variable temperatures and energy barriers for these intramolecular rate processes determined. The studies of the tyrosine resonances had been described in detail in a previous publication. The present paper describes the identification of the phenylalanine resonances and comments on some technical aspects which might be of quite general interest for the analysis of highly resolved 1H NMR spectra of proteins. Data for the tyrosines and the phenylalanines are compiled in three tables, i.e. the pK alpha-values for the tyrosines, the NMR parameters for all eight aromatics, and the parameters delta G not equal to, and, where available, delta H not equal to and delta S not equal to for the rotational motions of the rings.     

Catalytic activity of α-chymotrypsin in which histidine-57 has been methylated

The properties of a derivative of alpha-chymotrypsin in which histidine-57 has been methylated have been examined. Although the modified enzyme binds substrate with the same affinity as does native alpha-chymotrypsin, acylation and deacylation occur at much decreased rates. As for native alpha-chymotrypsin, a basic group of pK(a) approx. 7 is involved in both acylation and deacylation. The significance of these results is considered in relation to the normal function of histidine-57.     

NMR studies of carbonic anhydrase-fluorinated benzenesulfonamide complexes

Fluorine NMR has been used to examine complexes formed by 2-fluoro-, 3-fluoro-, and 2,5-difluorobenzenesulfonamide and human carbonic anhydrases I and II. The results show that all three inhibitors form complexes with both isozymes that have 2:1 inhibitor/enzyme stoichiometry. The fluorine spectra observed for all inhibitor-isozyme combinations are consistent either with rapid rotation of the aromatic ring of the inhibitor in the complexes or with preferential binding of only one of the two possible conformations of the inhibitors that are isomeric by virtue of rotation about the C1-C4 bond of the fluoro aromatic ring. Because ring rotation is slow in the case of the pentafluorobenzenesulfonamide-CA I complex, selective binding of rotamers is the explanation of these observations presently favored. A computer graphics study shows that formation of 2:1 complexes of CA I is feasible without appreciable distortion of the protein tertiary structure found in the crystalline state.     

Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water-miscible organic mixtures

We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.     

31P NMR of covalent phosphorylated derivatives of alpha-chymotrypsin

The structures of various covalent phosphorylated derivatives of alpha-chymotrypsin (alpha-CT) have been studied by 31P NMR spectroscopy. Diisopropylphosphoryl-alpha-chymotrypsin (alpha-DIPCT) shows a single 31P signal at ca. 0.0 ppm (pH 4). At low pH, the 31P NMR spectrum of alpha-DIPCT gradually changed with the appearance of one or two additional peaks. The ratio of the peaks varied with pH, time, and concentration. One of these two new downfield peaks (both at ca. 2.0 ppm) has been previously identified by Markley and co-workers (Markley, 1979; Porubcan et al., 1979) and van der Drift et al. (1985) as an aged monoisopropylphosphoryl-alpha-chymotrypsin (alpha-MIPCT) and is confirmed by our studies. A new additional downfield signal, separate from the alpha-MIPCT signal, is attributed to a dimer of the phosphorylated alpha-DIPCT. Phosphorylation of the enzyme with diphenyl chlorophosphate yields a monophenylphosphoryl-alpha-chymotrypsin (alpha-MPPCT) that also showed a single 31P signal at -2.1 ppm (pH 7). However, the spectrum did not change as a function of pH, incubation time, or concentration. Comparison of the 31P chemical shifts of the native and denatured phosphorylated derivatives of alpha-chymotrypsin suggests changes in the conformation about the P-O ester bonds are at least partially responsible for the various 31P chemical shift differences.     

Expression of functionality of alpha-chymotrypsin. An alternate binding mode in the substrate specificity site

Three-dimensional 2.8 A resolution x-ray crystallographic studies show that toluenesulfonamide and pipsylamide bind isomorphously in the aromatic specificity binding site of alpha-chymotrypsin. However, their orientation differs by about 90 degrees from that usually associated with substrate-like molecules, suggesting a nonproductive binding mode. A secondary binding site is also operative in one molecule of the dimer of the pipsylamide derivative and it is located some 22 A from the active site; however, this site is not operative in the toluenesulfonamide derivative. Binding of toluenesulfonamide and pipsylamide in the specificity site occurs without inducing any significant changes in the native enzyme structure, in contrast to the behavior observed upon tosylation or upon transition state analogue binding of phenylethaneboronic acid. The structural changes accompanying the formation of the latter derivatives are generally asymmetric with respect to the dimeric structure of alpha-chymotrypsin and are generally confined to the binding domain or cylinder 2 of the enzyme (sequence greater than 122). These changes are displayed in a new way via diagonal distance map representation.     

NMR studies of fluorinated serine protease inhibitors

Powers and co-workers have provided evidence that thiobenzyl N-heptafluorobutyrylanthranilate (I) is an extremely potent inhibitor of serine proteases, especially alpha-chymotrypsin (Teshima, T., Griffin, J. C., and Powers, J. C. (1982) J. Biol. Chem. 257, 5085-5091). We have prepared additional derivatives of this structure in which fluorine substitutions have been made on the aromatic rings and have attempted to carry out fluorine NMR studies of the interaction of Powers' compound and these new derivatives with chymotrypsin. The solubility of all inhibitors examined in solvent systems compatible with the retention of native enzyme structure is extremely low. While some nmr evidence for complex formation could be obtained, preparations of the complexes examined were metastable and precipitation of the inhibitor eventually limits the amount of complex that can be present in solution to such low levels that nmr experiments are impractical. An unusual effect of solvent composition on fluorine chemical shifts suggests that the conformation of the inhibitors in aqueous solution and when bound to the enzyme is different from that in organic solvents.     

Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.     

Unfolded, oxidized, and thermoinactivated forms of glyceraldehyde-3-phosphate dehydrogenase interact with the chaperonin GroEL in different ways

The interaction of GroEL with different denatured forms of glyceraldehyde-3-phosphate dehydrogenase* (GAPDH) has been investigated. GroEL does not prevent thermal denaturation of GAPDH, but effectively interacts with the thermodenatured enzyme, thus preventing the aggregation of denatured molecules. Binding of the thermodenatured GAPDH shifts the Tm value of the GroEL thermodenaturation curve by 3 degrees towards higher temperatures and increases the DeltaHcal value 1.44-fold, indicating a significant increase in the thermal stability of the resulting complex. GAPDH thermodenatured in the presence of GroEL cannot be reactivated by the addition of GroES, Mg2+, and ATP. In contrast, GAPDH denatured in guanidine hydrochloride (GAPDHden) is reactivated in the presence of GroEL, GroES, Mg2+, and ATP, yielding 11-15% of its original activity, while the spontaneous reactivation yields only 2-3%. The oxidation of GAPDH with hydrogen peroxide in the presence of 4 M guanidine hydrochloride results in the formation of the enzyme (GAPDHox) that cannot acquire its native conformation and binds to GroEL irreversibly. Binding of GAPDHox to one of the GroEL rings completely inhibits the GroEL-assisted reactivation of GAPDHden, but does not affect the GroEL-assisted reactivation of lactate dehydrogenase (LDH). The data suggest that LDH can be successfully reactivated due to the binding of the denatured molecules to the apical domain of the opposite GroEL ring with their subsequent release into the solution without encapsulation (trans-mechanism). In contrast, GAPDH requires the hydrophilic cavity for the reactivation (cis-mechanism).     

Studies on 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase(phe) from escherichia coli k12. 3. Structural studies.

1. Investigations with structural analogues of phenylalanine indicated an absolute requirement for the aromatic ring and both the alpha-carboxyl and alpha-amino groups of phenylalanine for inhibition of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) activity. Replacement of the alpha-H atom with a methyl group does not decrease the inhibition greatly. Varying degrees of inhibition were observed with o, m and p mono-substituted fluoro, chloro and hydroxy phenylalanines. D-Phenylalanine and several metabolites of the aromatic biosynthetic pathways do not inhibit enzymic activity. 2. Circular dichroism studies indicated that the native enzyme possesses approximately 26% alpha-helix. Both circular dichroic and ultraviolet difference spectra indicated that the addition of phenylalanine to the synthetase induces a conformational change involving a small alteration of the secondary structure and large alterations in th interactions of some of the aromatic residues of the enzyme. In particular, a tryptophan residue moves from an extremly hydrophobic environment to one less hydrophobic. 3. Kd for the binding of phenylalanine to the enzyme was determined spectrophotometrically to be 75 muM. 4. Chemical modification studies suggested that a sulphydryl group and possibly a lysine residue may be implicated in the catalytic activity of the enzyme.     

Specific recognition of single-stranded nucleic acids. Interaction of tryptophan-containing peptides with native, denatured, and ultraviolet-irradiated DNA

The binding of the tripeptide Lys-Trp-Lys to native, denatured, and ultraviolet-irradiated DNAs has been investigated by fluorescence spectroscopy. Two types of complexes are formed which both involve electrostatic interactions. Only one of them involves a stacking of the tryptophyl ring with nucleic acid bases. Quantitative analysis of fluorescence data shows that this stacking interaction is strongly favored in denatured as compared to native DNA. In ultraviolet-irradiated DNA, the peptide Lys-Trp-Lys binds selectively to unpaired regions around thymine dimers. Due to the stacking interaction of the aromatic amino acid with nucleic acid bases, this simple tripeptide is therefore able to discriminate between single-stranded and double-stranded regions in a nucleic acid.     

Hydrophobic clustering in nonnative states of a protein: interpretation of chemical shifts in NMR spectra of denatured states of lysozyme.

Chemical shifts of resonances of specific protons in the 1H NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross-links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, alpha-lactalbumin, in its partially folded "molten globule" state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.     

Synthesis and characterization of a selenium-containing substrate of alpha-chymotrypsin. Selenium-77 nuclear magnetic resonance observation of an acyl-alpha-chymotrypsin intermediate

The selenium-containing ester p-nitrophenyl (phenylselenyl)acetate, C6H5SeCH2C(O)-OC6H4-p-(NO2), has been synthesized, characterized as a substrate for alpha-chymotrypsin (k2/KM = 15.2 X 10(3) M-1 s-1, KMapp = 5.16 X 10(-6) M, pH 7.77, 33% CH3CN, 25 degrees C), and shown to be an active-site titrant for the enzyme. A synthesis of the selenium-77 enriched p-nitrophenyl (phenylselenyl)acetate in 53% yield from 94.4% elemental selenium-77, followed by its reaction with alpha-chymotrypsin (pH 5.0, 0-3 degrees C), permitted the observation of the (phenylselenyl)acetyl-alpha-chymotrypsin reaction intermediate by selenium-77 NMR spectroscopy. This acyl-enzyme species had a chemical shift of 275.1 ppm relative to dimethyl selenide. Accompanying this resonance was a lower intensity, pH-dependent resonance that is assigned to (phenylselenyl)acetate on the basis of a pH titration of the model compound. Deacylation in the presence of hydrazine sulfate produced a resonance at 332.3 ppm in addition to the 302.2 ppm resonance of (phenylselenyl)acetate at pH 7.85. Denaturation of the acyl-enzyme resulted in a shift of the 275.1 ppm resonance to 334.6 ppm at pH 4.90, in good agreement with the selenium-77 chemical shift of the model compound, methyl (phenylselenyl)acetate, in CDCl3 (333.3 ppm). The large shielding observed for the native acyl-enzyme in comparison to the denatured species can be attributed to a resonance-perturbed ester linkage and/or steric compression at a nonbonding orbital of the selenium nucleus.     

Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked.

The 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters. The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme. The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5. A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink.     

pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured alpha-chymotrypsin

A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of alpha-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with lambda(max) of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of alpha-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.     

Side-chain dynamics of two aromatic amino acids in pancreatic phospholipase A2 as studied by deuterium nuclear magnetic resonance

The flexibility of individual amino acid side chains of pancreatic phospholipase A2 in aqueous and micellar solutions was studied with deuterium nuclear magnetic resonance (2H NMR). Bovine pancreatic phospholipase A2 was selectively deuterated at the aromatic ring systems of Trp-3 and Phe-5 and porcine pancreatic phospholipase A2 at Trp-3 only. Solid-state 2H NMR spectra of the lyophilized enzymes exhibited quadrupole splittings on the order of 130 kHz, indicating almost complete immobilization of the aromatic ring systems. Exposure to a water-saturated atmosphere did not remove these steric constraints. However, side-chain mobility could be induced for the tryptophyl residue of the bovine enzyme by dissolving this enzyme in aqueous buffer or micellar solution whereas the phenyl ring always remained immobile and served as a probe for the protein's overall rotation. Typical correlation times for the tryptophyl and phenyl aromatic ring systems in aqueous solution were 7 ps and 13 ns (at 20 degrees C), respectively. The correlation time of the phenyl ring was longer than expected for the monomeric protein (approximately 6 ns), suggesting some aggregation of the protein at the high concentrations used for the NMR measurements. Addition of a micellar solution of oleoylphosphocholine had no influence on the motional freedom of the tryptophyl residue but approximately doubled the correlation time of the phenyl ring, indicating an increase of the effective volume of the tumbling particle due to lipid-protein interaction. A different behavior was observed for the Trp-3 residue of porcine phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)