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1、Protein Sequence DeterminationPRELIMINARY The basic strategy for sequencing a protein is to break the protein into fragments small enough to be sequenced directly. These sequences are spliced together by comparing the overlapping regions of the fragments. In this exploration, you will be guided thro
2、ugh each step using the protein ribonuclease A as an example. Denature Protein Before fragmenting the protein, it must be denatured; that is, it must be unfolded from its compact globular shape into an extended linear form to make it more accessible to attack by chemical and enzymatic reagents. Here
3、, each amino acid in the protein is drawn as a single sphere. Heating the protein or adding substances such as urea will denature most proteins. However, the resulting unfolded chain is not yet ready for analysis. Many proteins have disulfide cross-links between cysteine side chains, drawn here as y
4、ellow spheres. These must be cleaved to facilitate the sequence determination. Reduce Disulfides A common way to cleave disulfide bonds is to add the reducing agent 2-mercaptoethanol. Under suitable conditions, two of these mercaptoethanol molecules react to cleave a disulfide link in a polypeptide
5、by forming a disulfide bond between themselves. Effectively, the disulfide bond is transferred from the polypeptide to the mercaptoethanols. The newly liberated sulfhydryl groups must then be chemically protected to prevent the reformation of disulfide bonds through air oxidation. This is often done
6、 by treatment with iodoacetic acid, an alkylating agent that reacts with cysteine residues to form S-carboxymethylcysteine, a compound that is unreactive under the conditions to which the polypeptide will subsequently be exposed. Take a moment to examine the fully denatured protein. Convince yoursel
7、f that it is indeed a linear chain from one end to the other. When you are ready to continue, click on the NEXT button. Determine Subunits At this point, we must determine how many different types of subunits the protein we are dealing with contains. Since each peptide chain has only two ends, ident
8、ifying how many unique terminal groups there are will tell us how many nonidentical subunits we are dealing with. In a common technique, the N-terminal amino group is reacted with with dansyl chloride. The resulting dansyl polypeptide is then hydrolyzed under acidic conditions so as to completely de
9、grade it to its component amino acids with its N-terminal residue still a highly fluorescent dansylamino acid. Here the purple balls represent amino acids and the dansyl group is represented by a yellow ball that is covalently linked to the N-terminal residue. The protein has now been converted to a
10、 mixture of single amino acids, one or more of which are labeled with the dansyl group. The next step is to separate out the highly fluorescent dansyl-labeled residues and identify the amino acids to which they attached. This can be done by a variety of chromatographic techniques. Here we use an HPL
11、C apparatus set up specifically for amino acid separation and identification. The sample is injected and the dansyl-labeled residue (in yellow) separates from the others as it passes through the column. Its relative elution volume depends precisely on the amino acid to which it is attached, thereby
12、identifying the N-terminal residue. Here we detect only one dansylamino acid, indicating that our protein consists of only one type of polypeptide chain. More than one dansyl-labeled peak would have indicated that the protein consists of more than one type of polypeptide. These subunits would have t
13、o be separated from one another, usually by chromatographic means, before the subsequent steps of the analysis could be carried out. Of course, there is a slight chance that two different polypeptides have the same N-terminal amino acid, in which case the conclusion that there is only one polypeptid
14、e chain would be erroneous. ANALYZE SEGMENTS Cleave into Segments Polypeptide sequences longer than 40 to 100 residues cannot be directly sequenced and therefore polypeptides longer than this must be cleaved into manageable-sized fragments. Certain enzymes cleave peptide bonds with great specificity
15、. Trypsin, for example, will only hydrolyze peptide bonds after lysine or arginine residues if the preceeding residue is not proline. Here, the lysines are drawn in green and the arginines in blue. As trypsin locates the various lysine and arginine side chains, it hydrolyzes the following peptide bo
16、nd, releasing fragments from the polypeptide. These fragments have different lengths, but because trypsin cleaves all copies of the protein the same way, there are only a small number of unique fragments. In the case of ribonuclease A, trypsin digestion generates a total of 14 fragments. The mixture of fragments must now be separated. An HPLC device will again do the trick. The sample containing the trypsin-cleaved polypeptide fragments, here represented by purple
