Primary protein structure determination

The term "structural genomics" is used to describe how the primary sequence of amino acids in a protein relates to the function of that protein. Currently, the core of structural genomics is protein structure determination, primarily by X-ray crystallography, and the design of computer programs to predict protein fold structures for new proteins based on their amino acid sequences and structural principles derived from those proteins whose 3-dimensional structures have been determined. Plant natural product pathways are a unique source of information for the structural biologist in view of the almost endless catalytic diversity encountered in the various pathway enzymes, but based on a finite number of reaction types. Plants are combinatorial chemists par excellence, and understanding the principles that relate enzyme structure to function will open up unlimited possibilities for the... 

We define genomics as the study of DNA and the encoding process leading to protein formation. Proteomics is the study of proteins in a cell— identification of the entire protein complement of a cell, tissue, or organisms (the proteome). It includes the study of protein interaction and involves protein sequencing to determine the primary protein structure. 

The surface activity of proteins (as well as many of their other functions) depends on the so-called tertiary structure of protein molecules, which is determined by the spatial arrangement of their polypeptide chains. This tertiary molecular structure depends in turn on the primary protein structure - the aminoacid sequence, which is determined by the genetic code of a cell. The surface of a protein globule is mosaic-like it contains both polar... 

The primary protein structure is its unique sequence of a chain of amino acids (Campbell et al, 1999), called a polypeptide backbone. This sequence is determined by the genetic code of DNA. 

The efficiency of mass spectrometric methods in determining primary protein structure naturally leads to the question of their utility to characterize secondary, tertiary, and quaternary structures as well as the formation of noncovalent complexes. The success of mass spectrometry in approaching these problems is more limited. For example, there are some legitimate questions about the correspondence... 

Loo, J.A., Ogorzalek, R.R., and Andrewa, PC., 1993, Primary to Quartemary Protein Structure Determination with Electrospray Ionization and Magnetic Sector Mass Spectrometry, In Org.Mass Spectrom., 28, 1640-1649. 

If it is known that a drug must bind to a particular spot on a particular protein or nucleotide, then a drug can be tailor-made to bind at that site. This is often modeled computationally using any of several different techniques. Traditionally, the primary way of determining what compounds would be tested computationally was provided by the researcher s understanding of molecular interactions. A second method is the brute force testing of large numbers of compounds from a database of available structures. 

Different techniques give different and complementary information about protein structure. The primary structure is obtained by biochemical methods, either by direct determination of the amino acid sequence from the protein or indirectly, but more rapidly, from the nucleotide sequence of the... 

Whereas the primary structure of a protein is determined by the covalently linked amino acid residues in the polypeptide backbone, secondary and higher... 

The immunoglobulin structure in Figure 6.45 represents the confluence of all the details of protein structure that have been thus far discussed. As for all proteins, the primary structure determines other aspects of structure. There are numerous elements of secondary structure, including /3-sheets and tight turns. The tertiary structure consists of 12 distinct domains, and the protein adopts a heterotetrameric quaternary structure. To make matters more interesting, both intrasubunit and intersubunit disulfide linkages act to stabilize the discrete domains and to stabilize the tetramer itself. 

ACh was first proposed as a mediator of cellular function by Hunt in 1907, and in 1914 Dale [2] pointed out that its action closely mimicked the response of parasympathetic nerve stimulation (see Ch. 10). Loewi, in 1921, provided clear evidence for ACh release by nerve stimulation. Separate receptors that explained the variety of actions of ACh became apparent in Dale s early experiments [2]. The nicotinic ACh receptor was the first transmitter receptor to be purified and to have its primary structure determined [3, 4]. The primary structures of most subtypes of both nicotinic and muscarinic receptors, the cholinesterases (ChE), choline acetyltransferase (ChAT), the choline and ACh transporters have been ascertained. Three-dimensional structures for several of these proteins or surrogates within the same protein family are also known. 

Disulfide bridges are, of course, true covalent bonds (between the sulfurs of two cysteine side chains) and are thus considered part of the primary structure of a protein by most definitions. Experimentally they also belong there, since they can be determined as part of, or an extension of, an amino acid sequence determination. However, proteins normally can fold up correctly without or before disulfide formation, and those SS links appear to influence the structure more in the manner of secondary-structural elements, by providing local specificity and stabilization. Therefore, it seems appropriate to consider them here along with the other basic elements making up three-dimensional protein structure. 

Christian Anfinsen did the crucial experiment that showed that primary structure determines tertiary structure for proteins C. J. Epstein, R. F. Goldberger, and C. B. Anfinsen, Cold Spring Harbor Symp Quant Biol 28 439 (1963). This experiment is not always as simple... 

Even a small protein consists of many hundreds of atoms. The primary aim of a structure determination is to place each of these atoms in space by determining the coordinates of each atom relative to a fixed coordinate system. The only techniques that can provide detailed information of this type are based on diffraction methods, and X-ray diffraction is the primary method. We must consider the meaning of the structures derived from these studies. Now X-ray diffraction can only be used to determine structures in crystals. One is, however, really concerned with the structure of proteins in solution, and it is therefore necessary to examine the difference between structure in the solid state and the solution state. We consider differences in general between these two states, and then differences in specific cases. To perform structural studies in solution, spectroscopic methods must be used. These methods are quite different from diffraction methods, being concerned with specific absorption or emission... 

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