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Determining the Primary Structure of a Protein
Determining the sequence of amino acids in a protein is a routine, but not trivial, operation in classical biochemistry. Its several parts must be carried out carefully to obtain accurate results (Figure 5.14).
Step 1 in determining the primary structure of a protein is to establish which amino acids are present and in what proportions. Breaking a protein down to its component amino acids is relatively easy: heat a solution of the protein in acid, usually 6 M HCl, at 100°C to 110°C for 12 to 36 hours to hydrolyze the peptide bonds.
Separation and identification of the products are somewhat more difficult and are best done by an amino acid analyzer. This automated instrument gives both qualitative information about the identities of the amino acids present and quantitative information about the relative amounts of those amino acids. Not only does it analyze amino acids, but it also allows informed decisions to be made about which procedures to choose later in the sequenc-ing (see Steps 3 and 4 in Figure 5.14). An amino acid analyzer separates the mixture of amino acids either by ion-exchange chromatography or by high-performance liquid chromatography (HPLC), a chromatographic techniquethat allows high-resolution separations of many amino acids in a short time frame. Figure 5.15 shows a typical result of amino acid separation with this technique.
In Step 2, the identities of the N-terminal and C-terminal amino acids in a protein sequence are determined. This procedure is becoming less and less necessary as the sequencing of individual peptides improves, but it can be used to check whether a protein consists of one or two polypeptide chains.
In Steps 3 and 4, the protein is cleaved into smaller fragments, and the amino acid sequence is determined. Automated instruments can perform a stepwise modification starting from the N-terminal end, followed by cleav-age of each amino acid in the sequence and the subsequent identification of each modified amino acid as it is removed. This process is called the Edmandegradation.
The Edman degradation method becomes more difficult as the number of amino acids increases. In most proteins, the chain is more than 100 residues long. For sequencing, it is usually necessary to break a long polypeptide chain into fragments, ranging from 20 to 50 residues for reasons that will be explained later.
Proteins can be cleaved at specific sites by enzymes or by chemical reagents. The enzyme trypsin cleaves peptide bonds preferentially at amino acids that have positively charged R groups, such as lysine and arginine.
The cleavage takes place in such a way that the amino acid with the charged side chain ends up at the C-terminal end of one of the peptides produced by the reaction (Figure 5.16). The C-terminal amino acid of the original protein can be any one of the 20 amino acids and is not necessarily one at which cleavage takes place. A peptide can be automatically identified as the C-terminal end of the original chain if its C-terminal amino acid is not a site of cleavage.
Another enzyme, chymotrypsin, cleaves peptide bonds preferentially at the aromatic amino acids: tyrosine, tryptophan, and phenylalanine. The aromatic amino acid ends up at the C-terminal ends of the peptides produced by the reaction (Figure 5.17).
In the case of the chemical reagent cyanogen bromide (CNBr), the sites of cleavage are at internal methionine residues. The sulfur of the methionine reacts with the carbon of the cyanogen bromide to produce a homoserine lac-tone at the C-terminal end of the fragment (Figure 5.18).
The cleavage of a protein by any of these reagents produces a mixture of peptides, which are then separated by high-performance liquid chromatogra-phy. The use of several such reagents on different samples of a protein to be sequenced produces different mixtures. The sequences of a set of peptides produced by one reagent overlap the sequences produced by another reagent (Figure 5.19). As a result, the peptides can be arranged in the proper order after their own sequences have been determined.
The actual sequencing of each peptide produced by specific cleavage of a protein is accomplished by repeated application of the Edman degradation.
The sequence of a peptide containing 10 to 40 residues can be determined by this method in about 30 minutes using as little as 10 picomoles of material, with the range being based on the amount of purified fragment and the complexity of the sequence.
For example, proline is more difficult to sequence than serine because of its chemical reactivity. (The amino acid sequences of the individual peptides in Figure 5.19 are determined by the Edman method after the peptides are separated from one another.) The overlapping sequences of peptides produced by different reagents provide the key to solving the puzzle. The alignment of like sequences on different peptides makes deducing the overall sequence possible. The Edman method has become so efficient that it is no longer considered necessary to identify the N-terminal and C-terminal ends of a protein by chemical or enzymatic methods. While interpreting results, however, it is necessary to keep in mind that a protein may consist of more than one polypeptide chain.
In the sequencing of a peptide, the Edman reagent, phenyl isothiocyanate, reacts with the peptide’s N-terminal residue. The modified amino acid can be cleaved off, leaving the rest of the peptide intact, and can be detected as the phen-ylthiohydantoin derivative of the amino acid. The second amino acid of the original peptide can then be treated in the same way, as can the third. With an automated instrument called a sequencer (Figure 5.20), the process is repeated until the whole peptide is sequenced.
Another sequencing method uses the fact that the amino acid sequence of a protein reflects the base sequence of the DNA in the gene that coded for that protein. Using currently available methods, it is sometimes easier to obtain the sequence of the DNA than that of the protein. Using the genetic code, one can immediately determine the amino acid sequence of the protein. Convenient though this method may be, it does not determine the positions of disulfide bonds or detect amino acids, such as hydroxyproline, that are modi-fied after translation, nor does it take into account the extensive processing that occurs with eukaryotic genomes before the final protein is synthesized.
To finish this section, let’s go back to why we needed to cut the protein into pieces. Because the amino acid analyzer is giving us the sequence, it is easy to think that we could analyze a 100-amino-acid protein in one step with the ana-lyzer and get the sequence without having to digest the protein with trypsin, chymotrypsin, or other chemicals. However, we must consider the logistical reality of doing the Edman degradation. As shown in step 1 of Figure 5.20, we react the peptide with the Edman reagent, phenylisothiocyanate (PITC). The stoichiometry of this reaction is that one molecule of the peptide reacts with one molecule of PITC. This yields one molecule of the PTH derivative in step 3 that is then analyzed. Unfortunately, it is very difficult to get an exact stoichiometric match. For example, let’s say we are analyzing a peptide with the sequence Asp-Leu-Tyr, etc. For simplicity, assume we add 100 molecules of the peptide to 98 molecules of the PITC because we cannot measure the quantities perfectly accurately. What happens then? In step 1, the PITC is limiting, so we eventually end up with 98 PTH derivatives of aspartate, which are analyzed correctly and we know the N-terminus is aspartate. In the second round of the reaction, we add more PITC, but now there are two peptides; 98 of them begin with leucine and 2 of them begin with aspartate. When we analyze the PTH derivatives of round 2, we get two signals, one saying the derivative is leucine and the other saying aspartate. In round 2, the small amount of PTH derivative of aspartate does not interfere with our ability to recognize the true second amino acid. However, with every round, this situation gets worse and worse as more of the by-products show up. At some point, we get an analysis of the PTH derivatives that cannot be identified. For this reason, we have to start with smaller fragments so that we can analyze their sequences before the signal degrades.
The amino acid sequence of a protein can be determined using a multi-step process.
First, the protein is hydrolyzed into its constituent amino acids and the composition determined.
The protein is also cleaved into smaller fragments and these fragments are then sequenced by the Edman degradation.
By using overlapping fragments and the sequences determined, the sequence of the original protein can be deduced.
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