ROTATIONS OF TRYPTOPHAN RESIDUES IN PROTEINS

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W. & Jameson, D . M. (1986) Biophys. J . 49, 334a Vincent, M., de Foresta. B., Gallay, J. & Alfsen, A. (1982) Biochemis- try 21, 708-716 Wahl, Ph. (1969) Biochim. Biophys. Acfa 175, 55-64 Wahl, Ph. (1976) Biophys. Chem. 10, 91-104 Witholt, B. & Brand, L. (1970) Biochemistry 9, 1948-1956 Witholt, B. & Brand, L. (1973) in Fluorescence Techniques in Cell Biology (Thaer. A. A. & Sernetz, M., eds.), p. 283, Springer, New York Received 23 April 1986 Rotations of tryptophan residues in proteins ENRICO GRATTON, J. RICARDO ALCALA and GERARD MARRIOTT Departments of' Physics and Biochemistry. University of Illinois, Urhana. I1 61801, U . S . A . A classic study of the application of dynamic fluorescence depolarization methods to the investigation of protein inter- nal rotations was reported some years ago by Munro et al. (1979). The relatively large width of the excitation pulse used in those studies limited the time resolution to about 0.1 nanoseconds. Sommer et al. ( 1985) later measured the anisotropy decay of the single tyrosine residue in lima bean trypsin inhibitor using a femtosecond laser and a streak camera. More recently, Harris et al. (1986) have utilized time-correlated single photon counting with the narrow pulses obtained from a mode-locked laser to determine the rotational correlation times of the single tryptophan residue in mutants of T4 lysozyme. On the other hand, the use of differential phase and modulation ratio fluorometry to measure rotational properties of intrinsic fluorophores in proteins has been well established in our laboratory (Alcala et al., 1985; Gratton et al., 1985; Jameson et al., 1986; Prendergast et al., 1986). The high resolution, precision and selectivity of our apparatus enabled us to experimentally assess the theoretical findings of Karplus and co-workers (McCammon et al., 1977; Ichiye & Karplus, 1983) on the motions of tryptophan and tyrosine residues of some selected proteins, in particular lysozyme and bovine pan- creatic trypsin inhibitor (BPTI). The molecular dynamics calculations showed that rotational motions of those residues can occur in I-2Ops with an amplitude of several tens of degrees. This time range can be directly tested using the high-modulation frequencies now available in our pulsed-laser multifrequency phase fluorometer (Alcala et al., 1985). Materials and methods Lysozyme was from Boehringer-Mannheim and BPTI from Mobay Pharmaceuticals. Glycerol (gold-label), and p-terphenyl (gold-label) were purchased from Aldrich. Glycerol viscosities as a function of temperature were taken Abbreviation used: BPTI. bovine pancreatic trypsin inhibitor Vol. 14 from Segur (1953). All protein preparations were buffered in 50 mwsodium phosphate, pH 7.0. Lysozyme was chro- matographed o n Sephadex G-75 to remove higher aggre- gates. BPTI was determined to be pure by examination of the fluorescence emission spectrum which was almost superimposable on that of pure tyrosine. Excitation wavelength and emission filter (in parentheses) were as follows: 300 nm (Corning 0-52) for lysozyme and 287 nm (Triton X-100) for BPTI. Lifetime measurements were per- formed with the multifrequency phase fluorometer described by Alcala et al. (1985). This system uses a mode-locked argon-ion laser and a synchronously pumped dye laser (Spectra-Physics). U.V. radiation in the 285-3 10 nm region is obtained by doubling the output of the dye laser by an angle-tuned frequency doubler (Spectra-Physics). The modulation frequency set employed ranged from about 10 to 440 MHz. All lifetime measurements were obtained usingp-terphenyl in cyclohexane or methanol in the reference cell and with a polarizer set at 35' with respect to the vertical direction in the excitation beam. Differential phase and modulation ratio measurements were collected in the range used for lifetime data. Data analysis was performed using a non-linear least-squares routine. For the lifetime analysis a sum of exponentials was used. The following expression was used for the rotational analysis: where = 2 for the vertical component and /l = - I for the horizontal component of emission (excitation is vertical). r , ( t ) describes the anisotropy decay, I l c ( t ) the intensity decay, g,,, represent the pre-exponential factors for the anisotropy decay and R,,, the corresponding rotational correlation times. f; is the pre-exponential factor and t, the correspond- ing lifetime of the intensity decay part. The above equations are transformed to the frequency domain for the fit of the differential phase and modulation ratio data. The approxi- mate amplitude of the internal rotations was calculated as in Munro et ul. (1979) on the wobbling-in-cone model using = cos-'(+{[I 8(rK/r,,)''2]''2 - I}) the expression , @ where ro is the time zero anisotropy and rs is the value of the anisotropy when the internal rotations have equilibrated.