Folding pathway of guanidine-denatured disulfide-intact wild-type and mutant bovine pancreatic ribonuclease A

Dodge, Robert; Laity, John H.; Rothwarf, David M.; Shimotakahara, Sakurako; Scheraga, Harold A.

doi:10.1007/bf01901697

Cited by 23 publications

(34 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition to inhibiting the autocatalysis it could also accelerate folding if it binds already to refolding intermediates prior to the rate-limiting step of folding. An acceleration of folding by coenzymes or inhibitors was found for several proteins (33)(34)(35).…”

Section: Resultsmentioning

confidence: 99%

Autocatalytic Folding of the Folding Catalyst FKBP12

Schölz

Zarnt²,

Kern³

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Prolyl isomerases are folding enzymes and thus have the potential to catalyze their own folding. We show here that the folding of cytosolic FKBP12 (FK 506 binding protein) is an autocatalytic process both for the mature protein and for a fusion protein with an aminoterminal extension of 16 residues. Native FKBP contains seven trans-prolyl peptide bonds, and the cis-to-trans isomerizations of some or all of them constitute the slow, rate-limiting events in folding. The rate of an autocatalytic reaction increases with reactant concentration, because the product catalyzes its own formation. Accordingly, the folding of the fusion protein was more than 10-fold accelerated when the protein concentration was increased from 0.05 M to 10 M. At high concentrations of both forms of FKBP12 autocatalysis was very efficient, and the observed folding rate seemed to approach the rate of the fast direct folding reaction of the protein molecules with the correct (all trans) peptidyl-prolyl bond conformation.Protein folding in vitro and in vivo is often decelerated because it is coupled with prolyl isomerization (1-5). Cis/trans equilibria are usually established at the prolyl peptide bonds in an unfolded polypeptide chain, and, as a consequence, denatured proteins are heterogeneous mixtures of species which refold with different rates. Only the molecules with the nativelike prolines 1 can refold directly in a rapid reaction. The molecules with incorrect prolines refold in slow reactions that are limited in rate by the cis/trans isomerizations of these prolines. Proline-limited folding reactions are catalyzed in vitro and in vivo by peptidyl-prolyl cis/trans isomerases (3, 4, 6 -12). Four families of these ubiquitous proteins are now known (13-16), and their functions are not confined to catalyzing slow steps in protein folding (13,17 (24) and refolding can be followed by a change in protein fluorescence and by the regain of the prolyl isomerase activity.In an autocatalytic reaction the product accelerates its own formation and thus its rate is expected to increase with reactant concentration. For a proline-limited folding reaction, autocatalysis should thus lead to an increase in rate with protein concentration until the rate of direct fast refolding is approached. Therefore, in our refolding experiments, we varied the concentration of FKBP12 in a wide range, and in some folding experiments FK 506 was added to inhibit the prolyl isomerase activity of the refolded molecules. We found that autocatalysis contributes indeed to the refolding of most unfolded FKBP12 molecules. A strong autocatalytic rate enhancement was found in the folding of a variant of FKBP12 for which the direct and the proline-limited folding reactions differ vastly in rate. EXPERIMENTAL PROCEDURESMaterials-Urea (ultrapure) and guanidinium chloride (ultrapure) were from ICN Biomedicals, ␣-chymotrypsin and recombinant human cytoplasmic Cyp18 were from Boehringer-Mannheim, LiCl and CF 3 CH 2 OH were from Fluka, the assay peptide Suc-Ala-Phe-Pro-Phe-4-nitroanilide w...

show abstract

Section: Resultsmentioning

confidence: 99%

Autocatalytic Folding of the Folding Catalyst FKBP12

Schölz

Zarnt²,

Kern³

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Hence, no systematic error due to the fitting of the data has occurred. More details on the procedures used in this analysis can be found in Dodge et al (1994).…”

Section: Denton Et Almentioning

confidence: 99%

Kinetics of Folding of Guanidine-Denatured Hen Egg White Lysozyme and Carboxymethyl(Cys6,Cys127)-Lysozyme: A Stopped-Flow Absorbance and Fluorescence Study

Denton¹,

Rothwarf²,

Scheraga³

1994

Biochemistry

Self Cite

View full text Add to dashboard Cite

The folding kinetics of hen egg white lysozyme and of a three-disulfide derivative of lysozyme [carboxymethyl(Cys6,Cys127)-hen egg white lysozyme] have been studied by absorbance- and fluorescence-detected stopped-flow techniques. A "very-fast" phase with a time constant in the millisecond range has been observed by both absorbance and fluorescence when unfolded lysozyme in 4 M guanidine hydrochloride, 100 mM phosphate buffer, and pH 2.0 is refolded at 0.5 M guanidine hydrochloride, 100 mM phosphate, and pH 6.7. Data obtained from fluorescence-detected refolding studies show that a transient intermediate is formed during the very-fast refolding phase. This intermediate is characterized by substantial quenching of tryptophan fluorescence. In addition, analysis of the fluorescence data indicates the presence of an additional "burst" phase that occurs within the dead time of the instrument, < 3 ms. The very-fast phase is not observed during the refolding of the three-disulfide derivative. In addition, the three-disulfide derivative re-attains the final native folded conformation more rapidly than the unmodified protein over the range of temperatures studied (10-20 degrees C). We conclude that, not only does the presence of the disulfide bond between Cys6 and Cys127 slow down the overall folding process of lysozyme, but it also directs the folding of lysozyme through a pathway characterized by a non-native tertiary interaction(s).

show abstract

“…In a prolyl peptide bond, however, the trans isomer has only slightly lower enthalpy ( A H o = -1.27 kcal/mol) and equivalent entropy (Eberhardt et al, 1993). Further, isomerization of the Lys 41-Pro 42 peptide bond is of particular importance to RNase A, During protein folding, the isomerization of this bond is responsible for a slow kinetic phase , which is absent in P42A RNase A (Dodge et al, 1994).…”

Section: Contribution Of a Hydrogen Bond To Protein Stabilitymentioning

confidence: 99%

Contribution of a tyrosine side chain to ribonuclease A catalysis and stability–Contribution of Tyr 97 to RNase A catalysis and stability

et al. 1996

View full text Add to dashboard Cite

An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of kc,, being perturbed more significantly than that of K,,,. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidy1ic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and l l .7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr + Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond.

show abstract

Folding pathway of guanidine-denatured disulfide-intact wild-type and mutant bovine pancreatic ribonuclease A

Cited by 23 publications

References 45 publications

Autocatalytic Folding of the Folding Catalyst FKBP12

Autocatalytic Folding of the Folding Catalyst FKBP12

Kinetics of Folding of Guanidine-Denatured Hen Egg White Lysozyme and Carboxymethyl(Cys6,Cys127)-Lysozyme: A Stopped-Flow Absorbance and Fluorescence Study

Contribution of a tyrosine side chain to ribonuclease A catalysis and stability–Contribution of Tyr 97 to RNase A catalysis and stability

Contact Info

Product

Resources

About