Determining the sequence of amino acids

Tandem mass spectrometry has become an important tool for determining the sequence of amino acids in protonated peptides98 and the sequence of bases in deprotonated nucleic acids such as DNA.99 Despite the importance and widespread use of CID-MS to sequence peptides and nucleic acids, the mechanistic details of the dissociation processes are poorly understood. A better understanding of the... 

Genetic code Sequence of nucleotides along the DNA and coded in triplets (codons) along the mRNA that determines the sequence of amino acids in protein synthesis. The DNA sequence of a gene can be used to predict the mRNA sequence, and subsequently to predict the amino acid sequence. 

Here are the key points, (a) DNA is the stuff of genes, (b) DNA is a sequence of nucleotides, each of which carries one of four possible symbols, (c) DNA is orgaiuzed into a sequence of genes, (d) Genes determine the sequence of amino acids in proteins, (e) The sequence of amino acids determines the three-dimensional structure of proteins, which, in turn (f) determines their biological properties. These, in turn (g) determine the nature of the cell. It follows that the sequence of bases in DNA is the ultimate repository of the information required to specify the uifique biochemical personality of the cell. 

To figure out how genes work and how they direct the production of specific proteins that allow organisms to inherit traits, scientists started with the fact that chromosomes were known to be made up of protein and DNA. A series of experiments using bacteria and viruses that infect bacteria established that DNA, not protein, was the basic genetic material. Scientists figured out how DNA worked as the genetic material, how it was copied when a cell divided into two identical cells, and how DNA determined traits—that is, determined the sequence of amino acids in each protein that allow different traits to be expressed. 

DNA (deoxyribonucleic acid)—Carrier of genetic material that determines inheritance of traits. DNA is in chromosomes in every cell of the body except red blood cells and is copied when cells divide. DNA molecules are shaped like a double helix, and are composed of sequences of four bases adenosine (A), cytosine (C), guanine (G), and thymine (T). The sequence of the bases directs production of particular proteins by determining the sequence of amino acids in proteins. The double-helk structure of DNA helps it transmit genetic information. 

In the next step, translation, the sequence of nucleotides in the newly synthesized mRNA strand is used to determine the sequence of amino acids in the protein to be synthesized. This is done by way of a genetic code, which was fully deciphered by 1966 and is shown in Figure 13.34. According to the genetic code, it takes three mRNA nucleotides—each three-nucleotide unit is called a codon—to code for a single amino acid. The mRNA nucleotide sequence AGU, for example, codes for the amino acid serine, and AAG codes for lysine. (Note from Figure 13.34 that more than one codon can call for the same amino acid.) A few codons, such as AUG and UGA, are the signals for protein synthesis to either start or stop. 

Simplicity argues that the genetic blueprint specifying amino acid sequences in proteins should consist of consecutive, nonoverlapping triplets. This assumption turned out to be correct, as is illustrated by the DNA sequence for a gene shown in Fig. 5-5. In addition to the codons that determine the sequence of amino acids in the protein, there are stop codons that tell the ribosomal machinery when to terminate the polypeptide chain. One methionine codon serves as an initiation codon that marks the beginning of a polypeptide sequence. One of the valine codons sometimes functions in the same way. 

In 1950, when the study of ribosomes began, no methods for determining the sequences of amino acids in proteins or of nucleotides in nucleic acids existed.11 Sanger published the sequences of the two short chains of insulin in 1953, and the first transfer RNA sequence was published by Holley in 1965.21 Never-... 

One method for determining the sequence of amino acids in a large protein molecule involves treatment of the protein with Sanger s reagent, 2,4-dinitrofluoroben-zene. The reaction involves the -NH2 group at the end of the protein chain. Predict the product, and tell what kind of reaction is taking place. (See Sections 26.8-26.10.)... 

The sequence of bases in RNA determines the sequence of amino acids in a protein. Three bases code for a single amino acid for example, CAG is the code for glutamine. Therefore, a strand of RNA 2.73 X 10 bases long codes for a protein that has... 

The general strategy for determining the sequence of amino acids in a peptide or protein involves several steps. It is necessary (a) to hydrolyse the molecule completely and to determine quantitatively the relative molar proportions of amino acids present (Chapter 4) (b) to determine the molecular weight in order to calculate the number of residues of each amino acid present (c) to determine how many peptide chains are present and to separate these, bearing in mind that these may be... 

The first step in determining the sequence of amino acids in a peptide or a protein is to reduce any disulfide bridges in the peptide or protein. A commonly used reducing agent is 2-mercaptoethanol, which is oxidized to a disulfide. Reaction of the protein thiol groups with iodoacetic acid prevents the disulfide bridges from reforming as a result of oxidation by O2. 

Translation. The sequence of bases in mRNA determines the sequence of amino acids in a protein. 

Recently Edman (1950) described a method for determining the sequence of amino acids in a polypeptide chain, by splitting off one residue at a time starting from the A -terminal residue. The principle of the method is illustrated by the following equations ... 

Determining the sequence of amino acids in a protein is a routine, but not trivial, operation in classical biochemistry. It consists of several steps, which must be carried out carefully to obtain accurate results (Section 5.4). 

How does messenger RNA direct protein synthesis The sequence of bases in a given mRNA determines the sequence of amino acids in a specified protein. The size of mRNA molecules varies with the size of the protein. 

Table 21-1) to determine the sequence of amino acids in the polypeptide. The mRNA is AUG-CAU-UUG-UGU, which codes for the polypeptide Met-His-Leu-Cys. 

Biochemists are often interested in parameters that are not applicable for most classical analytes. Nucleic acids and proteins are both large biopolymers, consisting of sometimes thousands of monomers linked together. Methods are required to accurately determine the sequence of amino acids in a protein or the sequence... 

I) Purify and cleave the disulfide bridge (Section 9-10). (2) On a portion of the sample, degrade the entire chain by amide hydrolysis (6 N HCI. I IO°C. 24 h) to determine amino acid composition by using an amino acid analyzer. (3) On another portion of this material, apply repetitive Edman degradation to determine the sequence of amino acids. Because only nine are present, the entire chain can he sequenced in this way. 

However, there is no need to record all these product ion spectra because the complete amino acid sequence may frequently be deduced directly from the DADI/MIKE spectrum of the molecular ion. This has been utilized for the sequencing of polypeptides. As it is possible to determine the sequence of amino acids in oligopeptides even in a peptide mixture by DADI/MIKE spectrometry , the polypeptide is hydrolyzed to dipeptide subunits. The sequence of the different peptides is analyzed in the mixture and the data stored in a computer. In a second step, one single amino acid is hydrolytically split off from the original polypeptide. The... 

The genes determine the sequence of amino acids in a protein, but they do not, in and of themselves, determine how the protein is folded. Thus, the genes are only the foundation of cell activity, not the entire blueprint. 

In order to determine the sequence of amino acids in a peptide, initially it was subjected to partial hydrolysis. Thereby fragments were formed, smaller peptides the sequence of which could be more easily established. After micropreparative separation, e.g. by paper chromatography, the fragments were hydrolyzed and analyzed for amino acid composition both before and after treatment with nitrous acid. The amino add missing in the treated, desaminated, sample, was its N-terminal residue. The sequence of the whole peptide was then reconstructed through the appropriate combination of the structurally identified peptide fragments. A typical example, the eluddation of the structure of gramiddin S, is described on p. 205. 

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