Measurement of the refolding combination reaction between S-peptide and S-protein

Labhardt, Alexander M.; Ridge, John A.; Lindquist, Robert N.; Baldwin, Robert L.

doi:10.1021/bi00271a014

Cited by 25 publications

(17 citation statements)

References 21 publications

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“…Both pathways can contribute to exchange for every amide proton in RNase S. k 1 and k Ϫ1 are the rate constants for dissociation and association of S pep from S pro (40). k 1ex and k 2ex are the rate constants of exchange in the RNase S complex and dissociated fragments, respectively.…”

Section: Resultsmentioning

confidence: 99%

Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments

Chakshusmathi

Ratnaparkhi

Madhu

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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Ordered protein complexes are often formed from partially ordered fragments that are difficult to structurally characterize by conventional NMR and crystallographic techniques. We show that concentration-dependent hydrogen exchange studies of a fragment complex can provide structural information about the solution structures of the isolated fragments. This general methodology can be applied to any bimolecular or multimeric system. The experimental system used here consists of Ribonuclease S, a complex of two fragments of Ribonuclease A. Ribonuclease S and Ribonuclease A have identical three-dimensional structures but exhibit significant differences in their dynamics and stability. We show that the apparent large dynamic differences between Ribonuclease A and Ribonuclease S are caused by small amounts of free fragments in equilibrium with the folded complex, and that amide exchange rates in Ribonuclease S can be used to determine corresponding rates in the isolated fragments. The studies suggest that folded RNase A and the RNase S complex exhibit very similar dynamic behavior. Thus cleavage of a protein chain at a single site need not be accompanied by a large increase in f lexibility of the complex relative to that of the uncleaved protein.

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Section: Resultsmentioning

confidence: 99%

Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments

Chakshusmathi

Ratnaparkhi

Madhu

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…At the time the original exchange experiments were performed (Schreier & Baldwin, 1976;Rosa & Richards, 1981) the rate constants for association and dissociation of S protein from S peptide were not known and there was only limited information on the thermodynamics of binding of S peptide to S protein. The rate constants for association were determined subsequently by Labhardt et al (1983), and a complete thermodynamic characterization of the binding as a function of temperature was made recently by titration calorimetry . We have reanalyzed the proteolysis experiments in light of this fresh data and have also performed trypsin digestion studies of RNase A and RNase S as a function of protein concentration.…”

Section: Trypsin Digestion Studies Of Rnase a And Rnase Smentioning

confidence: 99%

Dynamics of ribonuclease A and ribonuclease S: Computational and experimental studies

et al. 1996

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RNase S is a complex consisting of two proteolytic fragments of RNase A: the S peptide (residues 1-20) and S protein (residues 21-124). RNase S and RNase A have very similar X-ray structures and enzymatic activities. Previous experiments have shown increased rates of hydrogen exchange and greater sensitivity to tryptic cleavage for RNase S relative to RNase A. It has therefore been asserted that the RNase S complex is considerably more dynamically flexible than RNase A. In the present study we examine the differences in the dynamics of RNase S and RNase A computationally, by MD simulations, and experimentally, using trypsin cleavage as a probe of dynamics. The fluctuations around the average solution structure during the simulation were analyzed by measuring the RMS deviation in coordinates. No significant differences between RNase S and RNase A dynamics were observed in the simulations. We were able to account for the apparent discrepancy between simulation and experiment by a simple model. According to this model, the experimentally observed differences in dynamics can be quantitatively explained by the small amounts of free S peptide and S protein that are present in equilibrium with the RNase S complex. Thus, folded RNase A and the RNase S complex have identical dynamic behavior, despite the presence of a break in polypeptide chain between residues 20 and 2 I in the latter molecule. This is in contrast to what has been widely believed for over 30 years about this important fragment complementation system. Keywords: dynamics; MD simulation; ribonuclease A: ribonuclease S; tryptic cleavage Fragment complementation systems are two or more fragments of a protein that can be reconstituted to form a complex with similar structure and activity to that of the native, uncleaved protein. Fragment complementation systems are a powerful tool in the study of protein folding and stability (Taniuchi et al., 1986). RNase S is one of the most well studied fragment complementation systems. It consists of S peptide (residues 1-20) and S protein (residues 21-124), two proteolytic fragments of the 124 amino acid protein RNase A. These fragments are obtained by cleavage of the peptide bond between Ala 20 and Ser 21 of RNase A by subtilisin. The two fragments can be reconstituted to give a noncovalently bound, active S peptide:S protein complex, RNase S (Richards & Vith-

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“…(ii) Refolding is more complex and opposing opinions exist. All workers agree that at least two unfolded populations exist, a 20% fast-folding and an 80% slow-folding fraction for ribonuclease A (9-23), ribonuclease S, and S-protein (24,25,(27)(28)(29). The kinetic characteristics of the slow-folding phase deviate from proline isomerism in model compounds.…”

mentioning

confidence: 92%

“…(i) During refolding, association of the S-peptide fragment with the S-protein fragment is one of the first detectable steps. In the I-state, the association rate kon = 104 M-sec -(310C) and the association constant Ka = 6 x 104 M-1 (320C) have been reported (27,30).…”

mentioning

confidence: 97%

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Kinetic circular dichroism shows that the S-peptide alpha-helix of ribonuclease S unfolds fast and refolds slowly.

Labhardt¹

1984

Proc. Natl. Acad. Sci. U.S.A.

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BiochemistryKinetic circular dichroism shows that the S-peptide a-helix of ribonuclease S unfolds fast and refolds slowly (folding pathway/fragment system/secondary structure/stopped flow/pH jump) A. M. LABHARDT Biozentrum der Universitaet Basel, Abteilung Biophysikalische Chemie, Klingelbergstrasse 70, Switzerland Communicated by Robert L. Baldwin, July 16, 1984 ABSTRACT It is shown that circular dichroism (CD) can distinguish between the S-peptide and the S-protein fragments of RNase S at 225 nm and 235 nm. The conformational source for the strong CD at 225 nm is the S-peptide a-helix. The structural assignment of the CD at 235 nm is not clear but it is shown to be largely due to the S-protein moiety. This situation is utilized to monitor the kinetics of pH-induced unfolding and refolding of the two moieties. It is observed that major changes occur both in the fast and slow phases of unfolding as well as refolding. Specifically, the S-peptide a-helix unzippering is a fast reaction, followed by slow kinetics only at 235 nm. These latter kinetics parallel the appearance of the slow-folding species commonly attributed to the accumulation of non-native proline isomers. In refolding, a large fraction of the CD of Sprotein at 235 nm recovers rapidly. The S-peptide a-helix zippers up last. These results are unexpected and their implications for the folding mechanism of ribonuclease are discussed.

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Measurement of the refolding combination reaction between S-peptide and S-protein

Cited by 25 publications

References 21 publications

Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments

Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments

Dynamics of ribonuclease A and ribonuclease S: Computational and experimental studies

Kinetic circular dichroism shows that the S-peptide alpha-helix of ribonuclease S unfolds fast and refolds slowly.

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