Konu Tarihi: 20.09.2010- 23:01

  

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N-TERMINAL PROTEIN SEQUENCING

A protein’s primary structure is the amino acid sequence of its polypeptide chains. The determination of the complete amino acid sequence of a protein is very important for the formulation of many concepts of biosciences for many reasons. First of all the knowledge of a protein’s amino acid sequence is essential for understanding its molecular mechanism of action and the function of the protein. Also sequence comparison ysis of proteins between different species gives information about the taxonomic homology and evolutionary relation of these species. Another important usage of amino acid sequence ysis data is clinical applications. Many inherited diseases are caused by mutations leading to an amino acid change in protein. With amino acid ysis the mutations can be determined easily. [1] Since determining amino acid sequence of a protein is very important many scientist worked on protein sequencing for many years and several methods for protein sequencing was developed. The methods of protein sequencing are basically divided into two groups: end group ysis and specific peptide cleavage. End group ysis methods are identification of the N-terminal or C-terminal amino acid of the protein. End group ysis can be made by using exopeptidases or some reagents that reacts with one terminal of the protein. In specific peptide cleavage reactions the endopeptidases are used. The peptides are cleavaged by specific endopeptidases from specific sites.
[1] There are several methods developed for N-terminal protein sequencing. One of these methods is “The Dansyl Method” which was developed by Gray and Hartley. In the Dansyl method, the reagent 1- diethylaminonaphthaene-5-sulfonyl chloride (dansyl chloride) reacts with primary amines of the peptides. Acid hydrolysis of the peptide yields a mixture of free amino acids plus the dansyl derivative. The dansyl amino acid is fluorescent under UV light and can be identified by thin-layer chromatography.
[2] Edman Degradation The most useful method for protein sequencing was developed by Pehr Edman in 1950s. Since 1950s “Edman Degradation” (Figure 1) is widely used in protein sequencing.
[3]. For Edman degradation phenylisothiocynate (PITC, Edman’s reagent) is used.
[4] The most important difference between Edman degradation and other methods is the amino acid sequence of a polypeptide chain from N-terminus is subjecting the polypeptide to repeated cycles of the Edman degradation and after each cycle identifying the N-terminal amino acid. [1] The Edman degradation is a series of chemical reactions that sequentially removes the N-terminal amino acids from a peptide or protein.
[5] Edman reactions occur in three main steps. In the first step (coupling) the N-terminal amino acid of the protein couples with PITC under alkali conditions to form PTC-protein. [3] In the second step (cleavage) the peptide bond of N-terminal PTC-protein undergoes acid cleavage and as a result of this reaction an unstable anilinothiazolinone-amino acid (ATZ-aa) derivative is produced. [3] In the last step (conversion) the unstable ATZ-aa is converted to phenylthiohydantoin-amino acid (PTH-aa) derivative. The PTC-aa is determined by HPLC chromatography. [3] Main Steps of Edman Degradation Step 1: Coupling The first step in Edman degradation is the coupling step (Figure 2). In this step first of all the protein is dissolved pyridine-water (1:1). The reaction temperature should be between 40-45 C. NaOH is added on the solution until the pH becomes 9.
[6] PITC (phenylisothiocynate) is added to the medium. Under these conditions the free amino terminus of the protein reacts with PITC. As a product of this reaction, a phenylthiocarbamyl - derivative of the amino terminal residue of the protein (PTC - protein) is produced.
[7] The by products of coupling reaction are removed by washing with n-heptane and ethyl acetate. PTC gives an exclusive reaction with the free amino groups of amino acids, and the reaction is same for all amino acids.
[7] A problem that can occur is the blockage of the protein. PITC can only react with the free amino group of the protein. Some times the N-terminal of the protein can be blocked by acetyl, acyl groups, or pyroglutamate residues formed by cyclization of glutamine or glutamate residues.
[9] This blockage prevents the coupling of the N-terminal of the protein with PITC.
[10] There were several methods developed for deblocking the blocked proteins for sequencing. But none of these methods have proven as efficient and applicable.
[10] Step 2: Cleavage In the second step PTC-protein is treated with an anhydrous acid like trifluoracetic acid (TFA), which results with the cleavage of the peptide bond between the first and the second amino acids of the protein.
[5] The products of this reaction are the anilinothiazolinone form of the terminal amino acid (ATZ - aa) and the “n-1” chain length protein. This protein again undergoes a coupling reaction in Edman cycle.
[7] Step 3 : Conversion In the conversion step, the unstable anilinothiazolinone - amino acid derivatives (ATZ - aa) are converted into a more stable phenylthiohydantoin - amino acid (PTH - aa) form. This reaction takes place at 64 C in the presence of trifluoracetic acid (TFA). The product of this reaction is PTH - aa.
[7] Step 4 : Identification of PTH - amino acid The PTH – amino acid is generally identified by reverse-phase HPLC (high performance liquid chromatography). [5] Using HPLC, each amino acid is identified by the elution time of the peak it produces on a chromatogram. [3] An example of HPLC chromatogram of PTH – aa’s is shown in Figure 5. N-terminal protein sequencing is performed by repetitive cycles of the four steps. But there are some limits for the number of cycles that can be performed in this way. The main factor that limits the number of cycles is the yielding chemicals produced during coupling and cleavage of each cycle. Also the side products that built up during the cycles prevent the detection of the true cleavage product. For this reasons, the successive cycle numbers is limited between 20-30. When longer peptide chains should be sequenced the protein first proteolytically digested and then the sequences of each fragment is determined.
[7] With modern instrumentation some computer-aided automated protein sequencers have been developed. Automated protein sequencers (Figure 6) allow setting up and control sequencing cycles, and collect the resulting data by specialized software. These sequencers automatically remove and yze amino acid residues from protein and peptide chains of various types and lengths. [11] Figure 6. Automated protein sequencer [11] References [1] Voet, D., Voet, J. G. (1995). Biochemistry. New York: John Wiley & Sons Inc. ch.6. p.106-115. [2] Walker, J. M. (1994). Basic Protein and Peptide Protocols. Totowa: Humana Press. ch.35. p.321-328. [3] Perkin-Elmer. (1995). Preparing Samples for Protein Sequencing. The Perkin-Elmer Corporation. [http://www.proteincentre.com/sample_prep.pdf]. ch.3. p.8-14. [4] White, A., Handler, P., Smith, E. L. (1964). Principles of Biochemistry. New York: McGraw-Hill Book Company. ch.9. p. 146-149, [5] reference 2. ch.36. p.329-334. [6] Edman, P. (1950). Method for determination of the amino acid sequence in peptides. ACTA Chemica Scandinavica, 4, 283-293. [7] Copeland, R. A. (1994). Methods for Protein ysis. New York: Chapman & Hall. ch.7. p. 146-149. [8] Cherry, J. P., Barford, R. A. (1988). Methods for Protein ysis. Ilionis: American Oil Chemists’ Society. ch.15. p.258. [9] Shannon, J., Beggerly, L., Fox, J. W. (1997). Guide to Protein Sequencing. [http://hsc.virginia.edu/research/biomolec/seqguid4.htm]. [10] reference 3. ch.8. p.41-413. [11] reference 3. ch.1. p.3.