Swine pepsinogen folding intermediates are highly structured, motile molecules

McPhie, Peter

doi:10.1021/bi00265a020

Cited by 24 publications

(11 citation statements)

References 32 publications

(46 reference statements)

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“…A fast phase could not be monitored by our manual procedure, but a slow phase having a first-order rate constant of 0.008 + 0.001 s-1 was observed (Fig. 8), which compares with the published value of 0.005+0.001 s-1 (McPhie, 1982). The amplitude of the slow phase increased with the time of exposure to urea; when [Urea] (M) Fig.…”

Section: Renaturation Of Prochymosinsupporting

confidence: 48%

“…Previous studies have shown that pepsinogen is fully able to regain its native conformation after renaturation from high concentrations of urea (McPhie, 1980(McPhie, , 1982. Prochymosin, Portions of stock protein were rapidly diluted into solutions of various urea concentrations and pH values, and the changes in fluorescence intensity were measured continuously.…”

Section: Renaturation Of Prochymosinmentioning

confidence: 99%

“…The refolding kinetics of pepsinogen are well characterized (McPhie, 1980(McPhie, , 1982 and involve two kinetic phases. A fast phase could not be monitored by our manual procedure, but a slow phase having a first-order rate constant of 0.008 + 0.001 s-1 was observed (Fig.…”

Section: Renaturation Of Prochymosinmentioning

confidence: 99%

“…We here present studies on the properties of prochymosin unfolded in urea, on the unfolding transition and its reversal, and on the kinetics of unfolding and refolding. Since the unfolding and refolding of prochymosin have not previously been studied, we have included, for comparison, experiments on the homologous zymogen pig pepsinogen, which has been investigated extensively in this respect (Perlmann, 1963;Frattali et al, 1965;McPhie, 1975McPhie, , 1980McPhie, , 1982Ahmad & McPhie, 1978). The work demonstrates that prochymosin and pepsinogen differ in their rates of unfolding and in the processes that they undergo when maintained in concentrated urea; whereas pepsinogen after unfolding in urea slowly converts into a slow-refolding form, prochymosin slowly converts into a form that cannot refold simply by dilution of the denaturant.…”

Section: Introductionmentioning

confidence: 99%

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Denaturation studies on natural and recombinant bovine prochymosin (prorennin)

Sugrue

Marston²,

Lowe³

et al. 1990

Biochemical Journal

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1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v. c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.

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Section: Renaturation Of Prochymosinsupporting

confidence: 48%

Section: Renaturation Of Prochymosinmentioning

confidence: 99%

Section: Renaturation Of Prochymosinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Denaturation studies on natural and recombinant bovine prochymosin (prorennin)

Sugrue

Marston²,

Lowe³

et al. 1990

Biochemical Journal

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“…We have also carried out experiments to evaluate the effect of X-Pro peptide bond cis-trans isomerization on the rate of refolding of the denatured enzyme. We used the double jump procedure, in which the protein is rapidly transformed into the unfolded state and then returned to native conditions through variable delay times (McPhie, 1982;Semisotnov et al, 1990). In more detail, renaturation of luciferase was initiated over 8-600 s delay times after fast unfolding in 8 M urea.…”

Section: Resultsmentioning

confidence: 99%

Folding of firefly luciferase during translation in a cell-free system.

1994

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In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell‐free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell‐free system while this protein is being translated from its mRNA. It is shown that ribosome‐bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half‐time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.

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Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

Röder¹,

Wüthrich²

1986

Proteins

127

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A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin (J. Mol. Biol. 135: 199-215, 1979), which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes.

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Swine pepsinogen folding intermediates are highly structured, motile molecules

Cited by 24 publications

References 32 publications

Denaturation studies on natural and recombinant bovine prochymosin (prorennin)

Denaturation studies on natural and recombinant bovine prochymosin (prorennin)

Folding of firefly luciferase during translation in a cell-free system.

Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

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