Molecular dynamics of tryptophan in ribonuclease-T1. II. Correlations with fluorescence.

The interactions of tryptophan-59 (TRP-59) and its protein environment in ribonuclease-T1 (RNAse-T1) were examined in a 50-ps molecular dynamics simulation. The simulation used was previously shown to demonstrate a fluorescence anisotropy decay that closely agreed with the experimentally determined limiting anisotropy for RNAse-T1 (Axelsen, P. H., C. Haydock, and F. G. Prendergast. 1988. Biophys. J. 54:249-258). Further characterization of TRP-59 side chain dynamics and its protein environment has now been completed and correlated to other photophysical properties of this protein. Angular fluctuations of the side chain occur at rates of 1-10 cycles/ps and are limited to +/- 0.3 radians in all directions. Side chain motions are primarily limited by nonpolar collisions, although most side chain atoms have some collisional contact with polar atoms in the adjacent protein matrix or water. The steric relationship between PRO-39 and TRP-59 changes abruptly at 16 ps into the simulation. Two types of interaction with water are observed. First, a structural water appears to H-bond with the greater than N-H group of TRP-59. Second, water frequently contacts the six-atom ring. The electrostatic field experienced by the TRP-59 rings appears to be relatively constant and featureless regardless of ring orientation. We make the following interferences from our data: The fluorescent emission of TRP-59 may be red-shifted relative to TRP in nonpolar solvents either as a result of specific interactions with the structural water or relaxations of proximal bulk water and polar protein moieties. The quenching efficiency of polar interactions with TRP-59 must be extremely low given their frequency and the high quantum yield of RNAse-T1. This low efficiency may be due to restricted and unfavorable interaction geometries. PRO-39 is located near two titratable HIS residues in RNAse-T1 and may be involved in pH-dependent fluorescence phenomena by virtue of a metastable interaction with TRP-59. The interaction of bulk water with TRP-59 illustrates features of the gated transition state model for transient exposure to exogenously added collisional quenching agents. The restrictive environment of TRP-59 suggests that extrinsic quenching can only occur via interactions with the edge of the indole six-atom ring and that the efficiency of a quencher in a protein environment is likely to be a function of molecular symmetry.     

Experimentally verifying molecular dynamics simulations through fluorescence anisotropy measurements

The fluorescence anisotropy decay of the single tryptophan residue in phospholipase A2 was studied by use of differential polarized phase fluorometry and computer simulations of protein dynamics. The results enable the verification of a simulated dynamic event by direct experimental measurement on the same time scale. When all hydrogen atoms are modeled explicitly, the simulations agree well with the experimental measurements. However, the measurements contradict simulations in which nonpolar hydrogens are incorporated into "extended" or "united" atoms. These simulations predict an anisotropy decay in excess of measured values and appear to seriously underestimate the electrostatic interactions occurring between water and aromatic side chains. The results support the general validity of studying protein dynamics with the molecular-mechanics approach and illustrate a potentially serious deficiency of simulations which do not explicitly model all hydrogen atoms.     

HIV-1 integrase catalytic core: molecular dynamics and simulated fluorescence decays.

Two molecular dynamics simulations have been carried out on the HIV-1 integrase catalytic core starting from fully determined crystal structures. During the first one, performed in the absence of divalent cation (6-ns long), the catalytic core took on two main conformations. The conformational transition occurs at approximately 3.4 ns. In contrast, during the second one, in the presence of Mg(2+) (4-ns long), there were no such changes. The molecular dynamics simulations were used to compute the fluorescence intensity decays emitted by the four tryptophan residues considered as the only chromophores. The decay was computed by following, frame by frame, the amount of chromophores that remained excited at a certain time after light absorption. The simulation took into account the quenching through electron transfer to the peptide bond and the fluorescence resonance energy transfer between the chromophores. The fit to the experimental intensity decays obtained at 5 degrees C and at 30 degrees C is very good. The fluorescence anisotropy decays were also simulated. Interestingly, the fit to the experimental anisotropy decay was excellent at 5 degrees C and rather poor at 30 degrees C. Various hypotheses such as dimerization and abnormal increase of uncorrelated internal motions are discussed.     

The binding site of sodium in the gramicidin A channel: comparison of molecular dynamics with solid-state NMR data.

The location of the main binding site for sodium in the gramicidin A (GA) channel was investigated with molecular dynamics simulations, using an atomic model of the channel embedded in a fully hydrated dimyristoyl phosphatidycholine (DMPC) bilayer. Twenty-four separate simulations in which a sodium was restrained at different locations along the channel axis were generated. The results are compared with carbonyl 13C chemical shift anisotropy solid-state NMR experimental data previously obtained with oriented GA:DMPC samples. Predictions are made for other solid-state NMR properties that could be observed experimentally. The combined information from experiment and simulation strongly suggests that the main binding sites for sodium are near the channel's mouth, approximately 9.2 A from the center of the dimer channel. The 13C chemical shift anisotropy of Leu10 is the most affected by the presence of a sodium ion in the binding site. In the binding site, the sodium ion is lying off-axis, making contact with two carbonyl oxygens and two single-file water molecules. The main channel ligand is provided by the carbonyl group of the Leu10-Trp11 peptide linkage, which exhibits the largest deviation from the ion-free channel structure. Transient contacts with the carbonyl group of Val8 and Trp15 are also present. The influence of the tryptophan side chains on the channel conductance is examined based on the current information about the binding site.     

Phosphorescence anisotropy of liver alcohol dehydrogenase in the crystalline state. Apparent glasslike rigidity of the coenzyme-binding domain

Phosphorescence anisotropy from internal tryptophan (Trp) residues in proteins which are in the crystalline state may provide an experimental approach suitable to study the flexibility of rather rigid segments of protein structure. The phosphorescence anisotropy of Trp-314 in liver alcohol dehydrogenase, which is enclosed within the beta-sheet forming the coenzyme-binding domain, was measured with the protein free in solution and in the crystalline state. In contrast to the free protein, where the rotational correlation time reflects the tumbling rate of the whole macromolecule, there is effectively no loss in anisotropy in the crystalline state. At room temperature, the triplet lifetime of 0.5 s implies that the rotational correlation time of the indole side chain must be larger than 1 s. Anisotropy data show that fluctuations of the indole ring about the average position can only be of limited amplitude (cone of semiangle less than 15 degrees) and that the resistance opposed by the beta-sheet to out-of-plane rotational motions is equivalent to a viscosity larger than 2.5 X 10(8) P, a value which confirms the particular rigidity anticipated for such an assembly of secondary structure.     

Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins

Measurements of the anisotropy of protein fluorescence as a function of an added collisional quencher, such as acrylamide, are used to construct Perrin plots. For single tryptophan containing proteins, such plots yield an apparent rotational correlation time for the depolarization process, which, in most cases, is approximately the value expected for Brownian rotation of the entire protein. Apparent limiting fluorescence anisotropy values, which range from 0.20 to 0.32 for the proteins studied, are also obtained from the Perrin plots. The lower values for the limiting anisotropy found for some proteins are interpreted as indicating the existence of relatively rapid, limited (within a cone of angle 0 degrees--30 degrees) motion of the tryptophan side chains that is independent of the overall rotation of the protein. Examples of the use of this fluorescence technique to study protein conformational changes are presented, including the monomer in equilibrium dimer equilibrium of beta-lactoglobulin, the monomer in equilibrium tetramer equilibrium of melittin, the N in equilibrium F transition of human serum albumin, and the induced change in the conformation of cod parvalbumin caused by the removal of Ca+2. Because multitryptophan-containing proteins have certain tryptophans that are accessible to solute quencher and others that are inaccessible, this method can be used to determine the steady state anisotropy of each class of tryptophan residues.     

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching

We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T ₁, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40°C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T ₁, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.Abbreviations Ac acrylamide - ACTH adrenocorticotropin hormone (1–24) - BPTI bovine pancreatic trypsin inhibitor - NATA N-acetyl-L-tryptophanamide - RNase T ₁ ribonuclease T ₁ - S. Nuclease staphylococcus aureus nuclease Supported by grants DMB-8804931 and DIR-8710401 from the National Science Foundation, and GM-39617 from the National Institutes of Health. J. R. Lakowicz acknowledges support from the Medical Biotechnology Center at the University of Maryland. I. Gryczynski was on leave from University of Gáansk, Institute of Experimental Physics, Gdansk, Poland, with partial support from CPBP 01.06.2.01 (Poland). H. Cherek was on leave from Nicholas Copernicus University, Torun, Poland, with partial support from CPBP 01.06.2.03 Offprint requests to: J. R. Lakowicz     

Solvent-exposed tryptophans probe the dynamics at protein surfaces.

The dynamics of single tryptophan (W) side chain of protease subtilisin Carlsberg (SC) and myelin basic protein (MBP) were used for probing the surface of these proteins. The W side chains are exposed to the solvent, as shown by the extent of quenching of their fluorescence by KI. Time-resolved fluorescence anisotropy measurements showed that the rotational motion of W is completely unhindered in the case of SC and partially hindered in the case of MBP. The rotational correlation time (phi) associated with the fast local motion of W did not scale linearly with the bulk solvent viscosity (eta) in glycerol-water mixtures. In contrast, phi values of either W side chains in the denatured proteins or the free W scaled almost linearly with eta, as expected by the Stokes-Einstein relationship. These results were interpreted as indicating specific partitioning of water at the surface of the proteins in glycerol-water mixtures.     

Raman spectra of chemically modified lysozyme. Oxindole derivatives.

The Raman spectra of oxidation products of lysozyme have been investigated. The protein was oxidized by N-bromosuccinimide and dimethyl sulfoxide/HCl. Depending on the experimental conditions one to six tryptophan residues are oxidized to oxindole. The most prominent difference between the spectra of lysozyme and its oxindole derivatives is the strong band at 1017 cm^?1 which displaces the tryptophan peak at 1010 cm^?1. Other tryptophan bands are also weakened corresponding to the number of the tryptophan side chains destroyed. Shifts are observed in the amide I and in the amide III regions sensitive to conformational changes. These shifts indicate conformational differences in the higher oxidized species and in the native enzyme, although the amide III maxima overlap with a strong oxindole band. Similar effects are observed in the range of the C-C stretching vibrations of the peptide backbone. If more than one tryptophan side chain is oxidized changes have also been found in the S-S stretching range. The evaluation of this effect is difficult because of the strong oxindole vibration appearing in this region. In species oxidized by great excess of N-bromosuccinimide the tyrosine vibrations can no longer be detected, indicating the modification of this amino acid too.     

Tryptophan dynamics of the FK506 binding protein: time-resolved fluorescence and simulations.

The FK506-binding protein (FKBP12) is important in the immunosuppressant action of FK506 and rapamycin. We have investigated Trp side chain dynamics in FKBP12, with and without a bound immunosuppressant, by measuring the Trp time-resolved fluorescence anisotropy decay r(t). The r(t) for W59 in aqueous uncomplexed FKBP12 at 20 degrees C is well described by a single exponential with a recovered initial anisotropy, r(eff)o, of 0.192 and an overall rotational correlation time for the protein, phi p, of 4.7 ns; r(eff)o = 0.214 and phi p = 4.2 ns for the FKBP12/FK506 complex. Using an expression for the order parameter squared, namely S2 = r(eff)o/rTo, where rTo is the vitrified steady-state excitation anisotropy, we recovered an S2 of 0.75 for W59 fluorescence in uncomplexed FKBP12 and S2 approximately equal to 1 in the FKBP12/FK506 complex. Results obtained for the FKBP12/rapamycin complex are similar to those found for the FKBP12/FK506 complex. Minimum perturbation mapping simulations were performed on the free and complexed forms of FKBP12 and the results were generally in agreement with the experimental data.     

A procedure for refining a coiled coil protein structure using x-ray fiber diffraction and modeling

We describe a combined use of experimental and simulation techniques to configure side chains in a coiled coil structure. As already demonstrated in a previous work, x-ray diffraction patterns from hard alpha-keratin fibers in the 5.15 A meridian zone reflect the global configuration of the chi(1) dihedral angle of the coiled coil side chains. Molecular simulations, such as energy minimization and molecular dynamics, and rotameric representation in the PDB, are used here on a heterodimeric coiled coil to investigate the dihedral angle distribution along the sequence. Different procedures have been used to build the structure, the quality assessment was based on the agreement between the simulated diffraction patterns and the experimental ones in the fingerprint region of coiled coils (5.15 A). The best one for building a realistic coiled coil structure consists of placing the side chains using molecular dynamics (MD) simulations, followed by side chain positioning using SMD or SCWRL procedures. The side chains and the backbone are equilibrated during the MD until they reach an equilibrium state for the t/g(+) ratio. Positioning the side chains on the resulting backbone, using the above procedures, gives rise to a well-defined 5.15 A meridian reflection.     

Fluorescence, CD, attenuated total reflectance (ATR) FTIR, and 13C NMR characterization of the structure and dynamics of synthetic melittin and melittin analogues in lipid environments.

The structure and dynamics of synthetic melittin (MLT) and MLT analogues bound to monomyristoylphosphatidylcholine micelles, dimyristoylphosphatidylcholine vesicles, and diacylphosphatidylcholine films have been investigated by fluorescence, CD, attenuated total reflectance (ATR) FTIR, and 13C NMR spectroscopy. All of these methods provide information about peptide secondary structure and/or about the environment of the single tryptophan side chain in these lipid environments. ATR-FTIR data provide additional information about the orientation of helical peptide segments with respect to the bilayer plane. Steady-state fluorescence anisotropy, fluorescence lifetime, and 13C NMR relaxation data are used in concert to provide quantitative information about the dynamics of a single 13C-labeled tryptophan side chain at position 19 in lipid-bound MLT, and at positions 17, 11, and 9, respectively, in lipid-bound MLT analogues. Peptide chain dynamics are probed by NMR relaxation studies of 13C alpha-labeled glycine incorporated into each of the MLT peptides at position 12. The cumulative structural and dynamic data are consistent with a model wherein the N-terminal alpha-helical segment of these peptides is oriented perpendicular to the bilayer plane. Correlation times for the lysolipid-peptide complexes provide evidence for binding of a single peptide monomer per micelle. A model for the membranolytic action of MLT and MLT-like peptides is proposed.     

Cyclic hexapeptides related to somatostatin. Conformational analysis employing 1H-NMR and molecular dynamics

We report the conformational analysis of a series of cyclic hexapeptides related to the hormone somatostatin utilizing 1H NMR spectroscopy and NOE restrained molecular dynamics. The conformational preferences and results from biological analysis of these analogs (previous paper) allow for refinement of the current understanding of the structure-activity relationship of somatostatin. For most of the molecules examined, a beta II' turn about the D-tryptophan-lysine residues, postulated to be required for biological activity, was present. From the NOE restrained molecular dynamics, it can be seen that the turn structure is important for the maintenance of the proper orientation of the side chains of the adjacent phenylalanine, tryptophan and lysine. The biologically active analogs have the side chains of lysine and D-tryptophan extended away from the 18-membered ring in close proximity to each other for a significant portion of the dynamic simulations. Although other conformations are accessible and monitored during the simulations, we believe this is important for biological recognition. The absence of the beta II' turn at the D-tryptophan-lysine disrupts this side chain array producing inactive molecules. The role of the bridging region, the Phe-Pro dipeptide, is to stabilize the beta II' turn and help maintain the proper orientation of the biologically important side chains.     

Site-specific tryptophan dynamics in class A amphipathic helical peptides at a phospholipid bilayer interface

The amphipathic helix plays a key role in many membrane-associating peptides and proteins. The dynamics of helices on membrane surfaces might be of importance to their function. The fluorescence anisotropy decay of tryptophan is a sensitive indicator of local, segmental, and global dynamics within a peptide or protein. We describe the use of frequency domain dynamic depolarization measurements to determine the site-specific tryptophan dynamics of single tryptophan amphipathic peptides bound to a phospholipid surface. The five 18-residue peptides studied are based on a class A amphipathic peptide that is known to associate at the interface of phospholipid bilayers. The peptides contain a single tryptophan located at positions 2, 3, 7, 12, or 14 in the sequence. Association of the peptides with egg phosphatidylcholine vesicles results in complex behavior of both the tryptophan intensity decay and the anisotropy decay. The anisotropy decays were biphasic and were fitted to an associated model where each lifetime component in the intensity decay is associated with a particular rotational correlation time from the anisotropy decay. In contrast, an unassociated model where all components of the intensity decay share common rotational modes was unable to provide an adequate fit to the data. Two correlation times were resolved from the associated analysis: one whose contribution to the anisotropy decay was dependent on the exposure of the tryptophan to the aqueous phase, and the other whose contribution reflected the position of the tryptophan in the sequence. The results are compared with existing x-ray structural data and molecular dynamics simulations of membrane-incorporated peptides.     

Synthesis and characterization of a sulfhydryl-reactive rhenium metal-ligand complex

We describe the synthesis and spectral characterization of a rhenium metal-ligand complex. This complex reacts with sulfhydryl groups via an iodoacetamide side chain on the phenanthroline ligand and displays a high limiting anisotropy near 0.35 when excited at 442 nm. When covalently linked to human serum albumin, this complex displayed a mean decay time of about 1 micros. This decay time is appropriate for measuring rotational correlation times on the microsecond time scale as may occur for large macromolecular complexes.     

Molecular contacts in the transmembrane c-subunit oligomer of F-ATPases identified by tryptophan substitution mutagenesis

When isolated in its monomeric form, subunit c of the proton transporting ATP synthase of Escherichia coli was shown to fold in a hairpin-like structure consisting of two hydrophobic membrane spanning helices and a short connecting hydrophilic loop. In the plasma membrane of Escherichia coli, however, about 9-12 c-subunit monomers form an oligomeric complex that functions in transmembrane proton conduction and in energy transduction to the catalytic F1 domain. The arrangement of the monomers and the molecular architecture of the complex were studied by tryptophan scanning mutagenesis and restrained MD simulations. Residues 12-24 of the N-terminal transmembrane segment of subunit c were individually substituted by the large and moderately hydrophobic tryptophan side chain. Effects on the activity of the mutant proteins were studied in selective growth experiments and various ATP synthase specific activity assays. The results identify potential intersubunit contacts and structurally non-distorted, accessible residues in the c-oligomer and add constraints to the arrangement of monomers in the oligomeric complex. Results from our mutagenesis experiments were interpreted in structural models of the c-oligomer that have been obtained by restrained MD simulations. Different stoichiometries and monomer orientations were applied in these calculations. A cylindrical complex consisting of 10 monomers that are arranged in two concentric rings with the N-terminal helices of the monomers located at the periphery shows the best match with the experimental data.     

Environments and conformations of tryptophan side chains of gramicidin A in phospholipid bilayers studied by Raman spectroscopy.

Raman spectroscopy has been used to investigate the hydrophobic interaction of the indole ring with the environments, the water accessibility to the N1H site, and the conformation about the C beta-C3 bond for the four tryptophan side chains of gramicidin A incorporated into phospholipid bilayers. Most of the tryptophan side chains of the head-to-head helical dimer transmembrane channel are strongly interacting with the lipid hydrocarbon chains, and the hydrophobic interactions for the rest increase with increasing hydrocarbon chain length of the lipid. One tryptophan side chain (probably Trp-15) is accessible to water molecules, another (Trp-9) is deeply buried in the bilayer and inaccessible, and the accessibilities of the remaining two (Trp-11 and Trp-13) depend on the bilayer thickness. The torsional angle about the C beta-C3 bond is found to be +/- 90 degrees for all the tryptophans irrespective of the membrane thickness. Binding of the sodium cation to the channel does not change the torsional angles but decreases the water accessibilities of two tryptophans (Trp-11 and Trp-13) considerably. In conjunction with a slight spectral change in the amide III region, it is suggested that the sodium binding causes a partial change in the main-chain conformation around Trp-11 and Trp-13, which results in the movements of these side chains toward the bilayer center. Two models consistent with the present Raman data are proposed for the tryptophan orientation in the dominant channel structure.     

Control of ditryptophan cross-linking: dihydrotryptophan as a tryptophan precursor in peptide synthesis.

In neat trifluoroacetic acid, tryptophan side chains cross-link to form a diastereomeric mixture of tryptophan dimers. Convergent oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) converts tryptophan dimers to ditryptophan. Since cross-link formation is under thermodynamic control, there has been no simple way of controlling the regiochemistry of the cross-linking process when more than one tryptophan side chain is present. Here, we show that dihydrotryptophan (Dht) can be incorporated into peptides as a tryptophan precursor, which reforms tryptophan upon treatment with DDQ. Dihydrotryptophan was prepared as a mixture of gammaS and gammaR diastereomers and the indoline nitrogen was protected with a Cbz group. The resulting amino acid, Nalpha-BOC-Dht(Cbz)-OH, was then incorporated into peptides as a mixture of diastereomers. Dht was resistant to tryptophan cross-linking in neat trifluoroacetic acid and was converted back to tryptophan during convergent oxidation of tryptophan dimers. While Dht is useful for control of ditryptophan regiochemistry and as a potential tryptophan analog, it is not a general strategy for Trp protection since DDQ is unlikely to be compatible with easily oxidized amino acids such as cysteine.     

Stereochemistry and mechanism of reactions catalyzed by indolyl-3-alkane alpha-hydroxylase.

The reaction of tryptamine with indolyl-3-alkane alpha-hydroxylase is shown to remove stereospecifically the pro-S hydrogen at C-2 of the side chain and to give hydroxytryptamine of "R" configuration. The reaction therefore proceeds stereospecifically with net inversion of configuration at C-2 of the tryptamine side chain. In the reaction of L-tryptophan methyl ester, the enzyme also catalyzes stereospecific removal of the pro-S hydrogen at C-3, but the product 3-hydroxytryptophan methyl ester is racemic at C-3. The unreacted tryptophan methyl ester is shown to incorporate solvent hydrogen into the pro-S position at C-3 in an at least partially stereospecific manner, suggesting that the reaction of L-tryptophan methyl ester is reversible. The hydrogens at C-1 of the tryptamine side chain and the alpha-hydrogen of L-tryptophan methyl ester are shown to be retained in the reactions. The results support the notion that the enzyme catalyzes stereospecific 1,4-dehydrogenation of 3-substituted indoles to the coresponding alkylidene indolenines as the primary reaction, followed by stereospecific or nonstereospecific hydration of these intermediates as a secondary process. Substrate specificity studies with a number of tryptophan analogs are in excellent agreement with such a mechanism.     

Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples. Applications to anisotropic rotations and to protein dynamics.

Enhanced resolution of rapid and complex anisotropy decays was obtained by measurement and analysis of data from progressively quenched samples. Collisional quenching by acrylamide was used to vary the mean decay time of indole or of the tryptophan fluorescence from melittin. Anisotropy decays were obtained from the frequency-response of the polarized emission at frequencies from 4 to 2,000 MHz. Quenching increases the fraction of the total emission, which occurs on the subnanosecond timescale, and thereby provides increased information on picosecond rotational motions or local motions in proteins. For monoexponential subnanosecond anisotropy decays, enhanced resolution is obtained by measurement of the most highly quenched samples. For complex anisotropy decays, such as those due to both local motions and overall protein rotational diffusion, superior resolution is obtained by simultaneous analysis of data from quenched and unquenched samples. We demonstrate that measurement of quenched samples greatly reduces the uncertainty of the 50-ps correlation time of indole in water at 20 degrees C, and allows resolution of the anisotropic rotation of indole with correlation times of 140 and 720 ps. The method was applied to melittin in the monomeric and tetrameric forms. With increased quenching, the anisotropy data showed decreasing contributions from overall protein rotation and increased contribution from picosecond tryptophan motions. The tryptophan residues in both the monomeric and the tetrameric forms of melittin displayed substantial local motions with correlation times near 0.16 and 0.06 ns, respectively. The amplitude of the local motion is twofold less in the tetramer. These highly resolved anisotropy decays should be valuable for comparison with molecular dynamics simulations of melittin.