Amino acids Protein structure determination

Partial Hydrolysis and Sequence Comparison In some cases it is also possible to determine the sequence of an unknown polypeptide by sequencing just a few of its amino acids and comparing this partial sequence with the database of known sequences for complete polypeptides or proteins. This procedure works if the unknown peptide turns out to be one that has been studied previously. (Studies of the expression of known proteins is one dimension of the field of proteomics. Section 24.14.) Due to the many sequence permutations that are theoretically possible and the uniqueness of a given proteins structure, a sequence of just 10—25 peptide residues is usually sufficient to generate data that match only one or a small number of known polypeptides. The partial sequence can be determined by the Edman method or by mass spectrometry. For example, the enzyme lysozyme with 129 amino acid residues (see Section 24.10) can be identified based on the sequence of just its first 15 amino acid residues. Structure determination based on comparison of sequences with computerized databases is part of the burgeoning field of bioinformatics. 

Biocatalysis refers to catalysis by enzymes. The enzyme may be introduced into the reaction in a purified isolated form or as a whole-cell micro-organism. Enzymes are highly complex proteins, typically made up of 100 to 400 amino acid units. The catalytic properties of an enzyme depend on the actual sequence of amino acids, which also determines its three-dimensional structure. In this respect the location of cysteine groups is particularly important since these form stable disulfide linkages, which hold the structure in place. This three-dimensional structure, whilst not directly involved in the catalysis, plays an important role by holding the active site or sites on the enzyme in the correct orientation to act as a catalyst. Some important aspects of enzyme catalysis, relevant to green chemistry, are summarized in Table 4.3. 

The molecular replacement method assumes similarity of the unknown structure to a known one. This is the most rapid method but requires the availability of a homologous protein s structure. The method relies on the observation that proteins which are similar in their amino acid sequence (homologous) will have very similar folding of their polypeptide chains. This method also relies on the use of Patterson functions. As the number of protein structure determinations increases rapidly, the molecular replacement method becomes extremely useful for determining protein phase angles. 

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... 

In 1972, Christian Anfinsen received the Nobei Prize in Chemistry for his principie of thermodynamic determinism, that the amino acid sequence aione determines the three-dimensionai structure. In the normal physiological state, the three-dimensional structure of a protein is the one in which the Gibbs free energy of the whole system is at a minimum. Many protein molecules have segments that are folded into a single-stranded a-helix, which is held together by the hydrogen bonds between coils, called a secondary structure. 

It has been our approach to protein structure determination by x-ray crystallography that it is imperative to begin with well-characterized and well-behaved protein. In particular, it is important that the protein be reasonably soluble and monodisperse in solution. Unfortunately, as discussed above, recombinant HIV-1 integrase satisfies neither of these conditions. One approach we and others have taken to circumvent these problems has been to examine truncated versions of HIV-1 integrase to determine if removal of amino acids from either terminus or both affects solubility and aggregation properties. 

The differences in primary structure can be especially informative. Each protein has a distinctive number and sequence of amino acid residues. As we shall see in Chapter 4, the primary structure of a protein determines how it folds up into a unique three-dimensional structure, and this in turn determines the function of the protein. Primary structure is the focus of the remainder of this chapter. We first consider empirical clues that amino acid sequence and protein function are closely linked, then describe how amino acid sequence is determined finally, we outline the many uses to which this information can be put. 

Thyrotropic Hormone. This hormone (TSH) stimulates the development of the thyroid and controls its secretion. Although purilied preparations of it have been obtained, they consist of a mixture of proteins of high mean molecular weight (about 30.000). Some of their amino acids have been determined, as well as their carbohydrates, bat the structures have not been elucidated. 

DT Blankenship, MA Krivanek, BL Ackerman, AD Cardin. High-sensitivity amino acid analysis by derivatization with o-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection applications in protein structure determination. Anal Biochem 178 227-232, 1989. 

Primary structure is the amino / ) acid sequence, which controls the shape of the protein and the role the protein serves in the body. Primary Structure Primary structure is the most fundamental of the four structural levels because it is the protein s amino acid sequence that determines its overall shape and function. So crucial is primary structure to function that the change of only one amino acid out of several hundred can drastically alter biological properties. The disease sickle-cell anemia, for example, is caused by a genetic defect in blood hemoglobin whereby valine is substituted for glutamic add at only one position in a chain of 146 amino acids. 

The primary structure of the ADH from L. brevis contains several structures which are typical for short-chain ADHs. The N-terminus, with a length of approximately 30 amino acids, is widely regarded as the coenzyme binding site with the conserved motif G-X-X-X-G-X-G, which is G-G-T-L-G-I-G for Lactobacillus brevis. A second conserved domain found in the L. brems-ADH sequence is a hydrophobic region comprising 10 or 11 residues, respectively. It contains two highly conserved glycines (G82 and G92), separated by nine amino acids. Such structures seem to be located inside the protein and determine the conformation of the enzyme. 

The sequencing strategy follows the order 1) preparation of pure protein, 2) selective cleavage of peptide bond 3) isolation of cleaved peptide fragments, 4) analysis of primary structure on amino acid sequencer, 5) determination of entire primary structure. Although this order has not changed since our primary structure determination work on AspAT carried out in early 1970, many improvements have been made, e.g. preparation of micro scale amount of sample and introduction of high sensitivity analytical instruments. These improvements have shortened analysis time and lowered the sample amount of required. 

Adair and Robinson (1) indicate that the refractive index of a protein or an amino acid is approximately determined by its elementary composition however, the structure of a molecule is also of importance. The values reported for amino acids are scattered and fragmentary (1, 10), and prior to our preliminary communication (25) no systematic investigation had accounted quantitatively for the relationship... 

Optical chirality of molecules is a characteristic measure of stereo-chemical property of biological, pharmaceutical, and metal coordination compounds. Choral structures of amino acids, proteins, DNAs, and various drugs in solutions have been determined from the measurement of circular dichroism (CD). However, small amount of molecules at the liquid-liquid interfaces has never been measured before CLM/CD method [19] and SHG/CD method have been reported [20],... 

The structure and properties of peptides and proteins depend critically upon the sequence of amino acids in the peptide chain. The first complete amino acid sequence of a protein, that of insulin (51 amino acid residues), was determined by F. Sanger in 1953. The process is now performed using automated protein sequencers, and involves step-by-step identification of amino acids at the N terminus of the protein using a chemical process known as Edman degradation. 

Fig. 5.3. Protein structure determination by X-ray diffraction. A. Crystals of porcine heart aconitase composed of 754 amino acids. The orthorhombic crystals shown are about 0.5 mm in the longest dimension. B. Film showing the diffraction pattern obtained from the above crystal. These data were used to obtain a 2.7 A resolution structure shown in two representations in panels C and D. Panel C shows the tracing of the protein backbone, with the small molecule (in red and yellow) in the central region depicting the iron-containing cofactor of the enzyme. Panel D shows the space-filling representation. (Courtesy of Dr Arthur H. Robbins, Miles Pharmaceuticals Inc. For details see A.H.Robbins and C.D.Stout (1989). Proteins Structure, Function, and Genetics 5, 289 312.)...

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