INVESTIGATION OF THE PHOTOINDUCED DISULFIDE DISRUPTION IN PROTEINS TERESA

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Investigation of the photo-induced disulfide disruption in proteins

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.

B INVESTIGATION OF THE PHOTOINDUCED DISULFIDE DISRUPTION IN PROTEINS TERESA ioinformatic 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.

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

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