Sorry, this site has been recently forced to change its server
and I am working hard on restroring the databse functionality, thank you for your patience. Tyrosine residues in different proteins:
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This page content:
1. Tyrosine is a common site of free radical formation in proteins
2. What does an EPR spectrum tell us about the radical's conformation? 2.1. Excel file-calculator download 3. About this database: linking EPR and structure 4. How to use the database; 4.1. Example: Human haemoglobin (HbA) reacting with hydrogen peroxide 4.1.1. How to determine q in the radical 4.1.2. How to find q in different Tyr residues 5. How to submit an entry to the database 5.1. How to masure angles f2 and f6 6. What's next? How this database will be developed 7. About this database and Contact |
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1. Tyrosine is a common site of free radical formation in proteins
Tyrosine is one of the twenty amino acids used as building blocks in proteins. It is a well established fact that free radicals can be formed on proteins, and tyrosine is certainly the most frequently reported site of such free radical formation. Many enzymes make use of the tyrosyl radicals (the products of one electron oxidation of tyrosine) in their catalytic mechanism. On the other hand, tyrosyl radicals can be harmful when formed in an uncontrolled fashion, e.g. when a peroxide reacts with haem proteins. When an electron is subtracted from tyrosine, a cation radical is formed which immediately (at physiological pH) drops the oxygen proton, so that the tyrosyl radical is a neutral species. The ring can rotate around the C1 - Cb bond in a free tyrosine (Fig . 1).
Therefore, depending on specific electrostatic environment, the rotational conformation of the phenol ring will be different in different tyrosine residues in proteins. 2. What does an EPR spectrum tell us about the radical's conformation?
EPR spectroscopy is the method of detection of paramagnetic species. Free radicals constitute a significant class of paramagnetic species since they, by definition, have a non-zero electron spin. If a radical lives long enough - its EPR spectrum can be recorded. Very often the EPR spectrum is so specific for a particular radical that it can be considered as its signature. This is not, however, the case for the tyrosyl radicals: the EPR spectra of the tyrosyl radicals found in different protein systems are remarkably variable. The main (though not exclusive) reason for that is the ability of the phenoxyl group to rotate around the C1 - Cb bond and the fact that the actual angle of such rotation is different in different tyrosyl radicals. The appearance of the EPR spectrum (its lineshape) is defined by the hyperfine interaction of the unpaired electron with the radical's protons. While such interaction for the four ring protons is independent of the orientation of the ring, on which the most of the spin density resides, the hyperfine splitting constants for the b-protons of the methylene group do depend on the rotation angle q according to the McConnell relation:
Thus, a simulation of an EPR spectrum of a tyrosyl radical provides us with the values of the isotropic hyperfine splitting constants for the methylene protons. These two values then can be used in system {2}, and the values of q and rC1 can be found. 2.1. Excel file-calculator download
The solutions of system {2} can be found numerically. I have created an Excel file (theta_in_tyr.xls) that can be used for solving system {2} on a routine basis:
This file can be used to perform two different tasks. You can find the values of rC1 and q for known values Ab1iso and Ab2iso. Alternatively, you can set some values for rC1 and q and find the values of the isotropic hyperfine constants Ab1iso and Ab2iso. I tried to make the theta_in_tyr.xls file user friendly. If, however, you have difficulties in using this file, please contact me and I will be happy to help. 3. About this database: linking EPR and structure
This database was created to store and to make easily accessible the information about rotational conformation of the ring in different tyrosines in different proteins. Why do we need such information?
As explained above, an EPR spectrum of a tyrosyl radicals in a protein provides very definite information
about the rotational angle q of the phenoxyl ring in the radical.
Successful simulation of such EPR spectrum yields two values of the isotropic hyperfine splitting
constants for the methylene protons, and angle q can be found from these
values. This database can then be used to identify the Tyr residues in the protein which are in the
conformation close to that found for the radical. Thus this database can help to assign the radical to
a particular Tyr site (and there could be dozens of those in one protein!).
4. How to use the database
First thing to do is to specify the protein structure file. Go to 'Search' on the top of this page, and you will find two ways of doing this. If you know the Protein Databank ID code of the protein (PDB ID), just select it from the pull-down menu and click 'Next'. Alternatively, you can search the records by a keyword. The screen that shows a search result for a protein will have a list of Tyr residues in a table. Ignore the f2 and f6 columns for the time being, you will only need to analysed the first three columns. The data in the table can be sorted by these columns. Since you probably want to know which Tyr residues are in a particular rotational conformation (the conformation found by EPR in the radical), you can set a value for q in the provided field (qtarget) and sort data in the table by the difference |q-qtarget|. The top of the table, when sorted by this difference, shows the Tyr residues with most close rotational conformation to that found in the radical by the EPR spectroscopy. 4.1. Example: Human haemoglobin (HbA) reacting with hydrogen peroxide
A tyrosyl free radical is formed in the reaction of metHbA with hydrogen peroxide. Before actually using the database, we will have to determine the rotational conformation of the phenoxyl ring in the radical. This can be done by simulation of the tyrosyl radical EPR spectrum. We have suggested a new algorithm for tyrosyl radical EPR spectra simulation (TRSSA) that works universally on all known lineshapes of tyrosyl radicals [Svistunenko D. A. & Cooper C. E. (2004) A new method of identifying the site of tyrosyl radicals in proteins, Biophys. J. 87: 582-595]. You might want to read this document which handles frequently asked question on TRSSA. 4.1.1. How to determine q in the radical
The EPR spectrum of the radical formed in the reaction of human haemoglobin with hydrogen peroxide and its simulation are shown in Fig. 3.
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