TOPICAL amino acid sequence

The last topic of this review is the prediction systems for the localization site of an input amino acid sequence. As stated previously, there are... 

An important topic of current research is how the sequence of amino acids in a newly synthesized protein can direct the folding of the chain into a precise, biologically active shape. Can the amino acid sequence be used to predict the final three-dimensional shape of the protein The short answer to this question is, Not completely, not yet. Present computer-aided predictions are about 70% accurate with... 

The primary level of structure in a protein is the linear sequence of amino acids as joined together by peptide bonds. This sequence is determined by the sequence of nucleotide bases in the gene encoding the protein (see Topic HI). Also included under primary structure is the location of any other covalent bonds. These are primarily disulfide bonds between cysteine residues that are adjacent in space but not in the linear amino acid sequence. These covalent cross-links between separate polypeptide chains or between different parts of the same chain are formed by the oxidation of the SH groups on cysteine residues that are juxtaposed in space (Fig. 4). The resulting disulfide is called a cystine residue. Disulfide bonds are often present in extracellular proteins, but are rarely found in intracellular proteins. Some proteins, such as collagen, have covalent cross-links formed between the side-chains of Lys residues (see Topic B5). 

Within the amino acid sequence there may be specific sequences which act as signals for the post-translational processing of the protein (e.g. glycosylation or proteolytic processing see Topic H5). 

The amino acid sequence data can be used to prepare antibodies specific for the protein of interest which can be used to study its structure and function (see Topic D5). 

The amino acid sequence can be used for designing DNA probes that are specific for the gene encoding the protein (see Topics 12 and 16). 

An example of an enzyme which has different isoenzyme forms is lactate dehydrogenase (LDH) which catalyzes the reversible conversion of pyruvate into lactate in the presence of the coenzyme NADH (see above). LDH is a tetramer of two different types of subunits, called H and M, which have small differences in amino acid sequence. The two subunits can combine randomly with each other, forming five isoenzymes that have the compositions H4, H3M, H2M2, HM3 and M4. The five isoenzymes can be resolved electrophoretically (see Topic B8). M subunits predominate in skeletal muscle and liver, whereas H subunits predominate in the heart. H4 and H3M isoenzymes are found predominantly in the heart and red blood cells H2M2 is found predominantly in the brain and kidney while HM3 and M4 are found predominantly in the liver and skeletal muscle. Thus, the isoenzyme pattern is characteristic of a particular tissue, a factor which is of immense diagnostic importance in medicine. Myocardial infarction, infectious hepatitis and muscle diseases involve cell death of the affected tissue, with release of the cell contents into the blood. As LDH is a soluble, cytosolic protein it is readily released in these conditions. Under normal circumstances there is little LDH in the blood. Therefore the pattern of LDH isoenzymes in the blood is indicative of the tissue that released the isoenzymes and so can be used to diagnose a condition, such as a myocardial infarction, and to monitor the progress of treatment. 

Monoclonal antibodies produced using this technology are now common tools in research because of their very high specificity. For example, they can be used to locate particular molecules within cells or particular amino acid sequences within proteins. If they are first bound to an insoluble matrix, they are also extremely useful for binding to and hence purifying the particular molecule from crude cell extracts or fractions (see Topic D5). They are also increasingly of use in medicine, both for diagnosis and as therapeutic tools, for example to inactivate bacterial toxins and to treat certain forms of cancer. 

Thanks to genomics, it is now much easier to deduce the amino acid sequence of a protein than by the older method of direct chemical analysis ( protein sequencing ). It is much harder to get a protein s three-dimensional structure than to get its sequence (i.e., essentially its chemical formula). The latter can be simply be deduced from the sequence of DNA, while the former is a major topic in other grand projects that will impact... 

Molecular modeling is also used in protein engineering to modify specifically or randomly selected residues of a protein to change the substrate specificity or to try to find an amino acid sequence that will fold into a specific, preselected 3D shape (the inverse protein folding problem). An introduction to this topic has been written by van Gunsteren. xhe principles of modeling have also been used to design new enzymes with altered substrate specificity. - ... 

As we have seen, metabolism and all the various other life processes are carried out by the action of enzymes, each one having its own unique amino acid sequence. Each type of cell will have many different enzymes, but on the other hand many enzymes present in one type of cell will be missing in another red blood cells, for example, do not have the enzymes of the Krebs cycle - they have no mitochondria to house them Even in a single tissue, different enzymes may be found at different times and in different physiological states as we mentioned in Topic 28, the PPP enzymes are only produced when required in biosynthetic tissues. So somewhere there has to be a set of instructions for making all these enzymes, and it must also be possible either to use the instructions or to ignore them as required. These instructions are called genes. 

Mathematical methods to compare nucleotide sequences have been reviewed by Waterman (9) and Davison (9a) the latter review is better suited for the uninitiated. The more recent topic of multiple nucleic acid sequence alignment has also been described by Waterman (10) as well as Sobel and Martinez (11). When coding regions are being compared use of the amino acid sequences results in much greater sensitivity as there are 20 amino and only four nucleic acids. Furthermore, several codons translate to one amino acid, amino acid mutation rates are better understood, and several characteristics exist to describe amino acids. However, Blaisdell (12) has recently discussed the use of preferences in nucleotide k-tuples to suggest relations in distant sequences even so, the problem of alignment still persists. 

Volume 13 contains chapters on various topics related to the structure and assembly of viruses, dealing in detail with nucleotide and amino acid sequences, as well as with particle morphology and assembly, and the structure of virus membranes and hybrid viruses. The first complete sequence of a viral RNA is represented as a multicolored foldout. 

The mutation rate fx of the nucleotide (or amino acid) at a sequence site is related to the popular notion of a molecular clock (Zuckerkandl and Pauling, 1965), because it determines after which time the clock ticks and anew mutation arises because of a copying error during meiosis. Whether this clock ticks uniformly is a topic of prolonged debate (summarized in Li, 1997). The question is usually treated by comparing sequence difference at (supposedly) neutral sites with evolutionary distance between species. 

Magnetic resonance techniques have again been popular for studying enzymes which are involved in phosphate hydrolysis and transfer. 31P or 19F N.m.r.1-2 and spinlabelling3 have all been used to study the interaction of substrates with these enzymes, while affinity labelling4 5 6 7 is another technique which has been used to obtain information about the sequence and conformation of amino-acid chains at the active sites of enzymes. Recently, these experimental methods have been applied to the study of cell membranes,6-7 and these are mentioned in a new series of books concerned with enzymes in biological membranes.8 A new journal, Trends in Biochemical Sciences, which contains concise, up-to-date reviews on these and other topics is published by Elsevier on behalf of the International Union of Biochemistry. 

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