Pancreatic RNase, folding

Probably not all proline residues are important for protein folding. Evidence for nonessential prolines came from a comparison of several homologous pancreatic RNases (Krebs et al., 1983, 1985) and cytochromes c (Babul et ai, 1978 Nall, 1990) that differ in the number of proline residues. Such prolines could be nonessential because they do not interfere with folding, or, alternatively, because they remain nativelike as regards isomeric state, after unfolding. 

Fig. 6. Acceleration of refolding of different proteins as a function of PPI activity. The acceleration factor is given as the ratio hlk of the observed rate constants for folding in the presence of PPI, k, and in the absence of PPI, ki,. The PPI activity is given as /f/ml. The K values are defined as described in footnote b to Table I. The following protein concentrations were used in the refolding experiments ( ) 2 /iM immunoglobulin light chain, ( ) 11 juAf porcine pancreatic RNase, and ( ) 17 fiM S-protein fragment of bovine RNase A. The final conditions for refolding were 0.25 M urea (0.30 M urea for porcine RNase), 0.1 Af Tris-HCl, pH 8.0, 10°C. Based on data from Lang el at. (1987).

In this section RNase A and RNase T1 are used as examples to illustrate the role of prolyl isomerizations for the unfolding and refolding of small single-domain proteins. Bovine pancreatic RNase A is selected because the history of the proline hypothesis and its experimental verification are closely related with this protein. The mechanism of RNase T1 folding is described because it is one of the major in vitro systems for investigating the function of prolyl isomerases as catalysts of proline-limited protein folding. 

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. ... 

Many secretory proteins—e. g., pancreatic ribonuclease (RNAse see p. 74)—contain several disulfide bonds that are only formed oxidatively from SH groups after translation. The eight cysteine residues of the RNAse can in principle form 105 different pairings, but only the combination of the four disulfide bonds shown on p. 75 provides active enzyme. Incorrect pairings can block further folding or lead to unstable or insoluble conformations. The enzyme protein disulfide iso-merase [1] accelerates the equilibration between paired and unpaired cysteine residues, so that incorrect pairs can be quickly split before the protein finds its final conformation. 

RNase A is a single-domain protein, a pancreatic enzyme which catalyses the cleavage of single-stranded RNA. This protein consists of 124 amino acid residues with a molecular mass of 13.7 kDa. It has traditionally served as a model for protein folding because it is small, stable and has a well-known native structure. The el protons of the four RNase A histidine residues are well-resolved from other protons in the H NMR spectrum of the native protein in D2O they have been used in this work to monitor the structural changes of four distinct segments in the molecule during cold, heat and pressure denaturation processes. His-12 and His-119 are part of the catalytic... 

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