Time-resolved fluorescence and anisotropy decay of the tryptophan in adrenocorticotropin-(1-24)

Abstract

The direct time-resolved fluorescence anisotropy of the single tryptophan residue in the polypeptide hormone adrenocorticotropin-(1-24) (ACTH) and the fluorescence decay kinetics of this residue (Trp-9) are reported. Two rotational correlation times are observed. One, occurring on the subnanosecond time scale, reflects the rotation of the indole ring, and the other, which extends into the nanosecond range, is dominated by the complex motions of the polypeptide chain. The fluorescence lifetimes of the single tryptophan in glucagon (Trp-25) and the 23-26 glucagon peptide were also measured. In all cases the fluorescence kinetics were satisfied by a double-exponential decay law. The fluorescence lifetimes of several tryptophan and indole derivatives and two tryptophan dipeptides were examined in order to interpret the kinetics. In close agreement with the findings of Szabo and Rayner [Szabo, A. G., & Rayner, D. M. (1980) J. Am. Chem. Soc. 102, 554-563], the tryptophan zwitterion exhibits emission wavelength dependent double-exponential decay kinetics. At 320 nm tau 1 = 3.2 ns and tau 2 = 0.8 ns, with alpha 1 = 0.7 and alpha 2 = 0.3. Above 380 nm only the 3.2-ns component is observed. By contrast the neutral derivative N-acetyltryptophanamide has a single exponential decay of 3.0 ns. The multiexponential decay kinetics of the polypeptides are discussed in terms of flexibility of the polypeptide chain and neighboring side-chain interactions.

Introduction of a Tryptophan Reporter Group into the ATP Binding Motif of the Escherichia coli UvrB Protein for the Study of Nucleotide Binding and Conformational Dynamics

“…39). The inten-sity of emission also increases in the native protein, slightly for Trp 47 and to a greater extent in Trp 51 . ; this study).…”

“…The multiple decay parameter values, presented in Table II, are suggestive of heterogeneity in either tryptophan environment, although the two shorter lifetimes contribute little to emission intensity (0.4 -1% for values of 0.11-0.15 ns, and 2-8% for ϭ 0.7 ns). Emission intensity is dominated in both proteins, more so in N51W, by a relatively long lifetime, 5.8 -6.4 ns, with intensity-weighted average lifetimes of 5.3 ns for Trp 47 and 6.1 ns for Trp 51 . Tryptophan in water, for comparison, has a lifetime of approximately 3 ns (3.0 ns for the peptidyl-Trp analog NATA; Ref.…”

“…UvrB* can interact with UvrA and form a preincision complex, but one that is less stable and with greatly reduced capacity for incision with added UvrC. Tryptophan (absent in the wild-type protein) has been introduced without significant alteration of function into the UvrB ATP binding motif, both at a site shown earlier to be mutable without phenotypic effect (Asn 51 ) and at a residue nearer the essential and conserved lysine (Lys 45 ) of the Walker motif (Phe 47 ). Both "reporter mutants" reveal conformational changes, localized to the binding site, that accompany proteolytic activation.…”

A long lifetime component in the tryptophan fluorescence of some proteins

“…When the ~L a state is excited at 300 nm, the fluorescence light is emitted in the range 330-360 nm. Hence, for these excitation and emission wavelengths one lifetime of the tryptophan fluorescence is expected and this is indeed found in some cases, the most notable one being N-acetyltryptophanamide (NATA) with a lifetime of about 3 ns (Szabo and Rayner 1980;Wijnaendts van Resandt et al 1980;Robbins et al 1980;Ross et al 1981;Chang et al 1983;Wagner et al 1987;Bismuto et al 1991;Vekshin et al 1992). However, the amino acid tryptophan already exhibits two lifetimes (at neutral pH).…”

Critical Amino Acids in the Transcriptional Activation Domain of the Herpesvirus Protein VP16 Are Solvent-exposed in Highly Mobile Protein Segments

“…The steady-state anisotropy (r ss ) values were also calculated using the lifetime instrument in the steady-state photon-counting mode. These small values, in the range of 0.059 -0.079, suggest that probes at either position were associated with fast segmental motion as seen in the flexible polypeptide adrenocorticotropin, where r ss ϭ 0.06 (50). By using "sum and difference" analysis, we found that these decays were best represented by two decay components.…”