Journal Article•
Intrinsic luminescence of protein as a tool for studying fast structural dynamics
TL;DR: The bathochromic fluorescence of several proteins evidences the slow (times much longer than 10 ns) reorientation mobility in these cases and 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.
Abstract: 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.
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TL;DR: Spectral shifts associated with red-edge excitation are observed for proteins emitting in the medium spectral range, suggesting the existence in these proteins of a distribution of microstates for tryptophan environment with various orientation of dipoles and of slow mobility of the field of these dipoles.
Abstract: 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.
86 citations
Cites background from "Intrinsic luminescence of protein a..."
...This served as a basis for classifying the azurin tryptophan residue as a representative of a very rare class A chromophore (Burstein 1977)....
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...1977) and of class S proteins (Burstein 1977) are unchanged....
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TL;DR: It has been revealed that the microenvironment of the tryptophan residue is in principle formed by non-polar hydrocarbon groups and the mechanism of the dramatic increase in fluorescence efficiency when the copper atom is removed has been discussed.
42 citations
TL;DR: A new model of the structure of OEP16 is suggested, suggesting that the protein is, in contrast to most other outer membrane proteins studied so far, purely alpha-helical, consisting of four transmembrane helices.
31 citations
TL;DR: A comparison of the emission spectra of wild-type C5 protein and some of its mutant derivatives is determined that the 318 nm maximum could be the result of a complex formed in the excited state as a result of hydrophobic interactions between Trp109, Phe18 and Phe73.
28 citations
Cites methods from "Intrinsic luminescence of protein a..."
...Therefore, it is not possible to classify the environment of Trp109 on the basis of the half-intensity width value of the emission spectrum (47.2 nm) according to the classi®cation system of Burstein (1977)....
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TL;DR: The experimental results revealed the unfolding of the native tetrameric enzyme in acidic pH range, causing exposure of the hydrophobic residues.
Abstract: 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
23 citations