Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay

Kofron, James L.; Kuzmič, Petr; Kishore, Vimal; Colon-Bonilla, Esther; Rich, Daniel H.

doi:10.1021/bi00239a007

Cited by 528 publications

(532 citation statements)

References 34 publications

(44 reference statements)

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“…The identification of the Pr55gag/Cyp18 interaction by these techniques established a quite high binding affinity of the proteins to each other. This finding is at variance with the high K,n values that could be separately determined, in part, for cis and trans oligopeptide substrates covering either the millimolar range for trans isomers or slightly lower for the cis peptides [19,34]. Attempts to saturate partially Cypl8 in the enzyme catalyzed refolding of denatured RNase TI were also unsuccessful even at the highest protein concentrations used [35].…”

Section: Discussionmentioning

confidence: 99%

“…The steady-state parameters became available by the improved protease coupled assay devised by Kofron et al [19]. The measurements were performed by monitoring the absorbance of released 4-nitroaniline at 390 nm (e = 11 814 M -1 cm -1) on a Hewlett Packard 8452A diode array spectrophotometer according to Schutkowski et al [20].…”

Section: Determination Of the Steady-state Parameters Kc~t And Km Of mentioning

confidence: 99%

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Extended binding sites of cyclophilin as revealed by the interaction with HIV‐1 Gag polyprotein derived oligopeptides

et al. 1996

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Oligopeptides derived from the gag polyprotein (Pr55 gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55 gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cypl8). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag 21s-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-I Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The ICs0 value of 184 ~M for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1--4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPlase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cypl8, and may add a previously unrecognized topological component to the known subsite specificity of cyciophilins.

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Section: Discussionmentioning

confidence: 99%

Section: Determination Of the Steady-state Parameters Kc~t And Km Of mentioning

confidence: 99%

Extended binding sites of cyclophilin as revealed by the interaction with HIV‐1 Gag polyprotein derived oligopeptides

et al. 1996

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“…During isomerization, the C-terminus of the peptide bond is free to rotate, whereas the N-terminus of the bond remains bound at the catalytic site of the enzyme (9). The authors also concluded that R55 is the key player in cis-trans isomerization, which is supported by mutational studies (9,10). Zhao and co-workers proposed the involvement of the guanidino group of the sidechain of R55 in hydrogen bonding with the prolyl nitrogen of the substrate.…”

Section: Introductionmentioning

confidence: 92%

“…Several efforts to understand the cis-trans isomerization of the peptidyl-prolyl bond have been made using different approaches such as NMR line-shape analysis, monitoring changes in R 2 upon substrate binding, crystal structure studies with different peptides, molecular dynamics simulations, and density functional theory calculations (9)(10)(11)(12)(13). Eisenmesser and co-workers proposed that the cis conformer of the peptide interacts with residues A101, N102, A103, K82, and R55 of the enzyme; afterward, the enzyme catalyzes rotation of the peptide-prolyl bond by 180 to produce the trans conformation and makes contacts around residues L98 and S99.…”

Section: Introductionmentioning

confidence: 99%

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Shukla

Singh

Vispute

et al. 2017

Biophysical Journal

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Cyclophilin catalyzes the ubiquitous process ''peptidyl-prolyl cis-trans isomerization,'' which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1 H-15 N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

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“…CyP possesses enzymatic activity for cis-trans isomerization of peptidyl-prolyl bonds and may catalyze protein folding (Fischer et al, 1989;Takahashi et al, 1989;Schonbrunner et a]., 1991). CsA is a potent inhibitor (Kj = lop9 to M) of the PPIase activity of CyP (Kofron et al, 1991). Although attention Abbreviations: hCyPA, human cyclophilin A; CyP, cyclophilin; CsA, cyclosporin A; PPIase, cis-trans peptidyl-prolyl isomerase; FKBP, FK506 binding protein; NFAT, nuclear factor from activated T-cells; IL, interleukin.…”

mentioning

confidence: 99%

Active site mutants of human cyclophilin A separate peptidyl‐prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition

et al. 1992

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Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--54, R55, F60, Q l l l , F113, and H126-have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry30, 2306-2310), H54Q, R55A, F60A, Q l l l A , F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q l l l A , F113A, and W121A retain 3-15% of the catalytic efficiency (kcoJKm) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.

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Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay

Cited by 528 publications

References 34 publications

Extended binding sites of cyclophilin as revealed by the interaction with HIV‐1 Gag polyprotein derived oligopeptides

Extended binding sites of cyclophilin as revealed by the interaction with HIV‐1 Gag polyprotein derived oligopeptides

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Active site mutants of human cyclophilin A separate peptidyl‐prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition

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