With the aim of finding non-equilibrium dipole-relaxational electronic excited states of tryptophan residues in proteins the dependence of the fluorescence emission maximum on excitation wavelength was studied for several proteins containing a single tryptophan residue per molecule. Spectral shifts upon red-edge excitation are not observed for short wavelength-emitting proteins (azurin, two-calcium form of whiting parvalbumin, ribonucleases C
2 and T
1). This may be because of the non-polar environment of the tryptophan residues in these proteins or because of the absence of dipole-orientational broadening of spectra. The effect was also not found for proteins emitting at long wavelengths (max. at 341–350 nm) —melittin at low ionic strength, IT-Aj1 protease inhibitor, myelin basic protein. In these proteins, the tryptophan residues are exposed to the rapidly relaxing aqueous solvent. Spectral shifts associated with red-edge excitation are observed for proteins emitting in the medium spectral range — human serum albumin in the N and F forms, IT-Aj1 protease inhibitor at pH 2.9, melittin at high ionic strength as well as the albumin-dodecylsulfate complex. This suggests the existence in these proteins of a distribution of microstates for tryptophan environment with various orientation of dipoles and of slow (on the nanosecond time scale) mobility of the field of these dipoles. As a result the emission proceeds from electronic excited states which are not at equilibrium.

Red-edge-excitation fluorescence spectroscopy of single-tryptophan proteins

The environment of the tryptophan residue in Pseudomonas aeruginosa azurin and its fluorescence properties

https://digitalcommons.uri.edu/cgi/viewcontent.cgi?article=1015&context=phys_facpubs

Folding Kinetics and Structure of OEP16

Fluorescence properties of a tryptophan residue in an aromatic core of the protein subunit of ribonuclease P from Escherichia coli

BACKGROUND

Tyrosinases are involved in enzymatic browning reactions in damaged fruits during post-harvest handling and processing. The overall structure of tyrosinase from Agaricus bisporus mushrooms at different pH values was monitored using fluorescence spectroscopy and molecular dynamics simulations.



RESULTS

When the pH value was increased from 6.0 to 9.0, the protein passed through several structural intermediates, including the tetramer, trimer and dimer stages. Changes in the secondary and tertiary structure of tyrosinase at neutral pH were outlined after running molecular dynamics simulations. A detailed check at the single-molecule level by means of molecular modeling tools suggested that the most important contribution to the fluorescence intensity is given by the H subunits with seven Trp and nine Tyr residues exposed to the solvent, whereas the lectin-like folded L subunits have only six Trp and three Tyr residues, of which only Trp15, Trp59 and Trp93 are partially exposed to the solvent.



CONCLUSIONS

The results indicated that the enzyme was sensitive to pH. The experimental results revealed the unfolding of the native tetrameric enzyme in acidic pH range, causing exposure of the hydrophobic residues. © 2014 Society of Chemical Industry

pH-induced structural changes of tyrosinase from Agaricus bisporus using fluorescence and in silico methods

The photoexcitation of Trp, Tyr or Phe chromophore in a protein is analogous to the instant (10(-15) to 10(-14) s) introduction of a local probe carrying strong electronic and protonic donor and acceptor moieties and, moreover, having a significant dipole moment (4 to 11 D). Depending on the protein structure mobility during the excited state lifetime, the intra-macromolecular complexes (the exciplexes) with polar groups can be formed and some reversible charge transfer processes, which qaench the fluorescence, can take place with greater or lesser probability. The shifts of Trp fluorescence spectra from 307 to 316 nm (due to the 1:1 exciplex formation) and to 325-330 nm (2:1 exciplex) are typical for many proteins. An additional spectral shift up to 350 nm is caused by the reorientational relaxation of a protein matrix dipoles during the nanosecond excitation lifetimes. The bathochromic fluorescence of several proteins evidences the slow (times much longer than 10 ns) reorientation mobility in these cases. The fluorescence quenching rates by proteic groups and extrinsic small quenchers is linearly related to the diffusion coefficient of a surrounding solvent. Analysis of such relationships allows, in principle, to evaluate the diffusional internal friction inside a protein and parameters of co-operativity and anisotropy of the mobility. Some sources of possible misinterpretations of Trp fluorescence depolarisation as a measure of the rotational mobility of indole.

E A Burshteĭn

Papers

Intrinsic luminescence of protein as a tool for studying fast structural dynamics