Ribonuclease amino acid sequences

Christian Anfinsen United States ribonuclease, amino acid sequencing and biological activity ... 

For each entry, a table summarizes information regarding (1) the X-ray diffraction data upon which the analysis is based, (2) the methods employed in structure analysis and refinement and their main results, and (3) some structural characteristics. In a separate table, the ribonuclease amino acid sequence is... 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

This classic experiment, carried out by Christian Anfinsen in the 1950s, provided the first evidence that the amino acid sequence of a polypeptide chain contains all the information required to fold the chain into its native, three-dimensional structure. Later, similar results were obtained using chemically synthesized, catalyti-cally active ribonuclease. This eliminated the possibility that some minor contaminant in Anfinsen s purified ribonuclease preparation might have contributed to the renaturation of the enzyme, thus dispelling any remaining doubt that this enzyme folds spontaneously. 

Various bacterial ribonucleases as well as the fungal ribonucleases Ty Uj, and U2 (see also Fig. 5-43) have amino acid sequences related to that of RNase A763 764 764a but with distinctly different three-dimensional structures. The active sites contain Glu, His, and Arg side chains. For RNase Ty Glu 58 and His 92 appear to provide acid-base catalysis with assistance from Tyr 38, Arg 77, and His 40.763 765 A glutamate carboxylate also appears to be the catalytic base in the related RNase, called barnase, from Bacillus amyloliquefaciens.766... 

ANFINSEN, CHRISTIAN B. (1916-1995). An American biochemist who won the Nobel prize for chemistry- in 1972 for his work on ribonuclease, especially concerning the connection between the amino acid sequence and die biologically active conformation. He shared the Nobel prize with Stanford Moore and William H. Stein. His doctorate was granted from Harvard. 

Thus it appears that the information for folding a protein to the native conformation is embodied in the amino acid sequence, because of the many possible disulfide-paired ribonuclease isomers, only one was formed in major yield. Further studies have given similar results with many other proteins. 

Ribonucleases are a widely distributed family of en-zymes that hydrolyze RNA by cutting the P—O ester bond attached to a ribose 5 carbon (fig. 8.12). A good representative of the family is the pancreatic enzyme ribonuclease A (RNase A), which is specific for a pyrimidine base (uracil or cytosine) on the 3 side of the phosphate bond that is cleaved. When the amino acid sequence of bovine RNase A was determined in 1960 by Stanford Moore and William Stein, it was the first enzyme and only the second protein to be sequenced. RNase A thus played an important role in the development of ideas about enzymatic catalysis. It was one of the first enzymes to have its three-dimensional structure elucidated by x-ray diffraction and was also the first to be synthesized completely from its amino acids. The synthetic protein proved to be enzymatically indistinguishable from the native enzyme. 

Identify the signal peptide and family signature sequences of pig pancreatic ribonuclease with the given amino acid sequence ... 

Figure 13.3. The Tree View is used to draw a phylogenetic tree from treefile of Phylip in radial, slanted cladogram, rectangular cladogram, or phytogram. The rectangular dado-gram for treefile derived from amino acid sequences of ribonucleases is drawn with TreeView.

The amino acid sequences of ribonuclease in Phylip format are given below. Perform phylogenetic analyses by Neighbor joining method using the Dayfoff PAM 001 matrix (default). 

Perform phylogenetic analysis by parsimony approach of ribonucleases with amino acid sequences given in Exercise 2. 

The hard-sphere potential has also been used to compute the sterically allowed conformations of small polypeptides of known amino acid sequence, viz. an octapeptide loop of ribonuclease (Nemethy and Scheraga, 1965) and the cyclic decapeptide gramicidin-S (Vanderkooi et al., 1966). We shall use the calculation for gramicidin-S to illustrate the method. This decapeptide consists of two identical pentapeptides joined into a ring by peptide bonds, the amino acid sequence being... 

Chemists have long appreciated that a protein s primary amino acid sequence determines its three-dimensional structure. It has also been known for some time that proteins are able to carry out their diversified functions only when they have folded up into compact three-dimensional structures. The protein-folding problem first gained prominence in the 1950s and 1960s, when Christian Anfinsen demonstrated that ribonuclease could be denatured (unfolded) and renatured reversibly. 

Fig. 19.2. Amino acid sequence of the enzyme ribonuclease Tj. Cysteine disulfide bridges which cross-link the linear polypeptide chain are indicated by heavy lines [593 a]...

How is the elaborate three-dimensional structure of proteins attained, and how is the three-dimensional structure related to the one-dimensional amino acid sequence information The classic work of Christian Anfinsen in the 1950s on the enzyme ribonuclease revealed the relation between the amino acid sequence of a protein and its conformation. Ribonuclease is a single polypeptide chain consisting of 124 amino acid residues cross-linked by four disulfide bonds (Figure 3.51). Anfmsen s plan was to destroy the three-dimensional structure of the enzyme and to then determine what conditions were required to restore the structure. 

Figure 3.51. Amino Acid Sequence of Bovine Ribonuclease. The four disulfide bonds are shown in color. [After C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 235 (1960) 633.]... 

Shuffle test. An enzyme that catalyzes disulfide-sulfhydryl exchange reactions, called protein disulfide isomerase (PDl), has been isolated. PDI rapidly converts inactive scrambled ribonuclease into enzymatically active ribonuclease. In contrast, insulin is rapidly inactivated by PDI. What does this important observation imply about the relation between the amino acid sequence of insulin and its three-dimensional structure ... 

---