Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans

Abstract

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.

Direct Kinetic Evidence for Triplet State Energy Transfer from Escherichia coli Alkaline Phosphatase Tryptophan 109 to Bound Terbium

“…Time-resolved phosphorescence measurements and decay analysis were made using instrumentation and methods described previously (27). Spectra were not corrected for photomultiplier wavelength response.…”

Stereoselective Binding of Flurbiprofen Enantiomers and their Methyl Esters to Human Serum Albumin Studied by Time‐Resolved Phosphorescence

“…d). Such multicomponent phosphorescence decays for single Trp proteins indicate the presence of various protein conformations that are stable on the phosphorescence timescale . Lifetimes of 2.7 and 0.4 ms were found at 0.2 M of KI.…”

Lack of correspondence between the room‐temperature phosphorescence decay‐components and Trp residues in a series of Trp→Cys or Trp→Phemutants of human carbonic anhydrase II

“…As discussed in the introduction, one would expect that these arise from distinct tryptophan residues, and that when these Trp residues are removed, the corresponding components of the decay would disappear. More specifically, based on previous experience with other proteins [8,17], we expected the longest decay component to be uniquely associated with the most buried, rigidly located Trp, i.e. W97.…”

“…8 ns) of light at 280 nm; an equal number of decays were collected form each mutant. Methods used for acquiring spectra, decays and subsequent analysis were as previously described [8].…”

“…Therefore in proteins containing more than one tryptophan the emission originating from solvent exposed tryptophans, typically with submillisecond life-times, is often obscured by the emission of rigidly disposed residues showing life-times up to two seconds. Single residue phosphorescence in multi-tryptophan containing proteins appears so far to be the rule rather than the exception (see references in [8]), and the residue responsible for the long-lifetime RTP is often assigned based on the locations of the tryptophans in the protein structure [9]. If the RTP decay components can be assigned to specific residues, and especially in proteins where the RTP is originating from a single residue, this technique can serve as a very specific probe for the local structure around the emitting tryptophan.…”