Investigation of the photo-induced disulfide disruption in proteins
Teresa Neves Petersen, Søren Klitgaard, Esben Skovsen, Steffen Petersen
Background
The amino acids in proteins have different properties, and the aromatic amino acids are of
particular interest since these are able to absorb ultraviolet photons, and upon excitation
these aromatic fluorophores can re-emit the energy as a new photon. However, the
excited state energy can instead be transferred to nearby acceptors. Another interesting
amino acid residue is cysteine, which is capable of binding to another cysteine in the
protein polymer, thus forming a disulfide bridge. Disulfide bridges provide enhanced
stability to the 3-dimensional structure of a protein.
When analysing the amino acid composition around disulfide bonds one finds the
aromatic amino acids as preferred spatial neighbours. This is interesting since it is also
known that illumination of aromatic amino acids, especially tryptophan, can cause a
disruption of nearby disulfide bridges. One of the causes of the disruption of the disulfide
bond is the transfer of an electron from the excited aromatic residue to the disulfide bond,
which is then reduced. The ejected electron, upon salvation, can be detected with
transient absorption spectroscopy (they absorb light around 700nm).
Other residues in the vicinity would also play a role to either promote or inhibit this
electron transfer depending on their charge.
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Power 0.14mW
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SS
UV
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Trp
Power 0.1mW
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Relative orientations of the two sulfur
atoms in disulfide in various proteins
Power 0.014mW
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Fluorescence intensity (counts/s)
Bioinformatic tools can be used to predict from a 3D protein model, a disulfide bond’s
susceptibility to break upon illumination would be useful e.g. light-induced
immobilisation.
Fitted experimental
kinetic traces (grey)
with our model’s
(solid lines) equation.
Time (s)
Plots for a range of proteins showing the occurrence of different groups of amino acids in a sphere of an
8Å radius around a tryptophan neighboring a disulfide bond. Red and yellow means under-represented
while blue and green means occuring more often than what should be expected.
Project description
The project will include database mining (structural information), and visualisation
software will be used to investigate the local environment around structural triads of
aromatic residues and cystines. Further steady state experiments will be conducted in
order to experimentally determine how factors such as pH and temperature affect the
disulfide bond disruption. If time allows pump-probe spectroscopy can be involved in
order to determine reaction kinetics of intermediate species in the reaction.
Proposed strategy / methods
The students will need to do following to achieve the goals in the project:
- Investigate a number of Protein Data Bank files in order to identify the location of
other amino acid residues that can affect the charge transfer.
- Investigate the relative orientation of aromatic amino acids and cystines in order
to determine the efficiency of dipole-dipole interactions.
- To predict whether pH changes could affect the efficiency of the disruption and to
test the hypothesis experimentally
- To determine the concentration of thiol groups formed upon UV illumination of
selected proteins
- To monitor the ultrafast processes (fs, ps timescales) associated with light induced
reaction: monitoring the formation of solvated electrons, ionic and radical species
formed upon UV illumination of protein samples. The lifetime of this species will
also be monitored. This work will be done at the “Ultrafast Biospectroscopy Laser
Lab” installed in the clean room.
References
- M. T. Neves-Petersen, P H. Jonson, and S. B. Petersen (1999), Amino acid neighbours
and detailed conformational analysis of cysteines in proteins, Protein Engineering 12 (7),
535-548
- M. T. Neves-Petersen, Z. Gryczynski, J. Lakowicz, P. Fojan, S. Pedersen, E. Petersen,
and S. B. Petersen (2002), High probability of disrupting a disulphide bridge mediated by
an endogenous excited tryptophan residue, Protein Science 11, 588-600
- J. R. Lakowicz (1999), Principles of Fluorescence Spectroscopy, 2nd Ed. Kluwer
Academic/Plenum Publishers, New York
- D. V. Bent, and E. Hayon (1975), Excited state chemistry of aromatic amino acids and
related peptides. III. Tryptophan, Journal of the American Chemical Society 97 (10)
- Y. Chen, and M. D. Barkley (1998). Toward understanding tryptophan fluorescence in
proteins, Biochemistry 37, 9976-9982
- J. J. Prompers, C. W. Hilbers, and H. A. M. Pepermans (1999), Tryptophan mediated
photoreduction of disulphide bonds causes unusual fluorescence behaviour of Fusarium
solani pisi cutinase. FEBS Lett. 45(6), 409-416.
- P. R. Callis, and T. Liu (2004), Quantitative prediction of fluorescence quantum yields
for tryptophan in proteins, J. Phys. Chem. B 108 4248-4259