Protein folding RNase

The studies of RNase Aand cytochrome c (Qi etal., 1998 Sosnick etal., 1997) show that caution is required in interpreting burst phenomena in protein folding. However, they do not require a reinterpretation of the cases in which the molten globule character of the burst-intermediate has been established (Arai and Kuwajima, 2000 Chamberlain and Marqusee, 2000). 

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. 

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. 

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... 

Emission spei a ot RNase A with the trp 92 insertion are shown in Rgure 16.52. The excitation wavelengfo was 280 nm. so foat b< osine and tryptophan are exched. The surprising feature of these spectra is foat foe tyrosine contribution is Inghest in foe native protein. This is the opposite of what is observed for most proteins. The reason for this unusual result is quenching, in foe folded state, of trp-92 by foe nearby aspartate residue. Hence, trp-92 b this engme ed protein provides a sensitive probe of protein folding, and its intensity increases nearly 100-fold when RNase A is unfolded. 

In order to analyze the irradiated complexes for reaction products among proteins and ribosomal RNA, proteins are extracted with acetic acid following the method of Hardy et al. From a reaction mixture irradiated at 13° for 3 hr and digested with RNase as described above, 200-j l aliquots are shaken at 4 for 40 min after addition of 400 /d of acetic acid and 20 /J of 1 Af MgClj. After centrifugation (10 min, 20,000 g), the supernatant liquid is plated on GF/A filters. Macromolecular material is precipitated with 10% trichloroacetic acid at 4°. The rRNA pellet is dissolved in 100 / 1 of 150 roM NaCl, 50 mJf EDTA pH 7.0, 0.5% SDS, and precipitated on GF/A filters with 10% trichloroacetic acid as described above. The results (Table II) show that iV-acyl-Phe-tRNA is covalently bound to rRNA rather than to ribosomal proteins. When RNase digestion is carried out as described above but with a 10-fold increased quantity of RNase A (= 1 ftg/100 fil), no radioactivity is found in the rRNA fraction, indicating hydrolysis of the reaction product. 

There are many examples of the stabilization of the native structure by specific ligands, substrates, or effectors (see Yon, 1969 Citri, 1973). However, addition of these ligands at early stages of protein folding may induce conformations that are different from the native one. For example, reoxidation of RNase in the presence of various nucleotides gives conformational isomers with altered functional properties (Gutte, 1978). 

Plausibly, proline isomerization plays a role in protein folding (Freedman, 1979), and certain prolines probably have to be in the right configuration before the protein achieves its native structure. Some other proline are permissive. Although serious evidence has been provided that proline isomerization accounts for the slow kinetic phase in the case of RNase, nothing permits one to generalize this interpretation to other proteins. Even, when... 

In a detailed kinetic study of recombination of S peptide with S protein during the refolding of RNase S, Labhardt and Baldwin (1979a,b) found that the combination of the two fragments occurs before folding of S protein. Furthermore, RNase S is formed more rapidly than S protein alone. However, S protein gives a stable folding intermediate at low temperatures and at pH 1.7 but not at pH 6.8. 

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