Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters

Moses, Edwin; Hinz, Hans‐Jürgen

doi:10.1016/s0022-2836(83)80130-2

Cited by 80 publications

(55 citation statements)

References 36 publications

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“…The culture medium we used for the production of type B neurotoxin [Proteose-peptone 2%; yeast extract 1% (both from Difco); ϪZ Amine type B (Sheffield Chem, Norwich, NY, USA) 1%; Na-thioglycolate (Sigma) 0.05%; glucose 1%] contained trypsin inhibitory activity (inhibition of BAPNA hydrolysis), even after autoclaving. The pancreatic trypsin inhibitor is well known for its thermal stability (Gebhard et al, 1986;Moses and Hinz, 1983). We independently confirmed the reported thermostability: 500 mg bovine pancreatic trypsin inhibitor (Sigma) was added to 100 ml of C. botulinum type E culture medium; the trypsin inhibitory activity of this solution after autoclaving 15 min at 121°C was about 90% that of what it was before autoclaving.…”

Section: Resultssupporting

confidence: 74%

Partial characterization of a 36-kDa protein from Clostridium botulinum type E that inhibits trypsin and chymotrypsin.

Prabakaran

DasGupta

1999

J. Gen. Appl. Microbiol.

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Botulinum neurotoxin serotypes (A to G) are produced as ϳ150-kDa single-chain proteins by certain strains of Clostridium botulinum, C. butyricum, and C. baratii that are grouped as proteolytic, also called phenotypic group I, and nonproteolytic, or phenotypic group II (Collins and East, 1998;Hatheway, 1993;Minton, 1995). Protease(s) endogenous to the bacteria cleave (nick) peptide bond(s) between the two conserved Cys residues of the single-chain neurotoxin. The proteolytically processed (nicked) neurotoxin, a dichain protein, is more toxic than its precursor singlechain protein. The neurotoxin serotype A harvested after sufficient incubation (e.g., 96 h) of the proteolytic culture is found as the dichain protein, and earlier harvests yield only single-chain or a mixture of single and dichain molecules (Dekleva and DasGupta, 1989), and the neurotoxin serotype E remains in the singlechain form because the culture lacks the endogenous protease(s) (DasGupta and Rasmussen, 1983;DasGupta and Sugiyama, 1972a, b). The type B neurotoxin, produced by the proteolytic strain, harvested even after 96 h is a mixture of Ն90% single-chain and Յ10% dichain molecules (DasGupta and Sugiyama, 1976;Kozaki and Sakaguchi, 1975). One could speculate that a protease inhibitor produced by the organism inhibits the protease(s) responsible for nicking of type B neurotoxin and thus limits the endogenous single to dichain conversion (DasGupta and Sugiyama, 1976). Indeed, Höyem and Skulberg (1962) had reported the presence of a trypsin-inhibiting activity in the supernatants of cultures of C. botulinum that produced neurotoxin serotypes A, B, and E and noted maximum inhibitory activity in the serotype B culture. They partially characterized the inhibitor only from type E culture; it was easily dialyzable and heat stable (activity was not lost after 1-2 h at 100°C or 15 min autoclaving at 120°C). No other report on this trypsin inhibitor is apparent since its first report (Höyem and Skulberg, 1962).We report purification to homogeneity of a trypsin inhibitor formed in C. botulinum type E culture and Nterminal amino acid sequence of the ϳ36-kDa inhibitory protein. The protein inhibited both trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). To our knowledge, this novel polypeptide has not been reported in any protein data bank and is the first polypeptide protease inhibitor of this large range isolated from a bacterial culture. We find it interesting that the culture phenotypically nonproteolytic produces this protease inhibitor. Materials and MethodsStock culture of C. botulinum type E (strain Alaska E-43 stored at Ϫ80°C) was grown (day 1) in cooked meat medium (Difco, Detroit, MI), at 30°C (Ϯ1°C). On day 2, 13-L glass carboys (Pyrex), each containing 9,500 ml of media (pH 7.3), were autoclaved for 1 h; then to each was added glucose (100 g in 500 ml water) separately autoclaved for 30 min. The final composition of the culture medium was yeast extract

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Section: Resultssupporting

confidence: 74%

Partial characterization of a 36-kDa protein from Clostridium botulinum type E that inhibits trypsin and chymotrypsin.

Prabakaran

DasGupta

1999

J. Gen. Appl. Microbiol.

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“…This approximation is reasonable, as the thermal stability of BPTI is high. 51 The test protein, trp-cage, is modeled without these constraints and thus free to fold and unfold in the simulations. The assumed backbone conformation of BPTI is a model approximation of the crystal structure (PDB ID 4PTI), derived by Monte Carlo with minimization.…”

Section: A Modelmentioning

confidence: 99%

Equilibrium simulation of trp-cage in the presence of protein crowders

Bille

Linse

Mohanty

et al. 2015

The Journal of Chemical Physics

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Link to publication Citation for published version (APA): Bille, A., Linse, B., Mohanty, S., & Irbäck, A. (2015). Equilibrium simulation of trp-cage in the presence of protein crowders. Journal of Chemical Physics, 143(17), [175102]. DOI: 10.1063/1.4934997General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. While steric crowders tend to stabilize globular proteins, it has been found that protein crowders can have an either stabilizing or destabilizing effect, where a destabilization may arise from nonspecific attractive interactions between the test protein and the crowders. Here, we use Monte Carlo replicaexchange methods to explore the equilibrium behavior of the miniprotein trp-cage in the presence of protein crowders. Our results suggest that the surrounding crowders prevent trp-cage from adopting its global native fold, while giving rise to a stabilization of its main secondary-structure element, an α-helix. With the crowding agent used (bovine pancreatic trypsin inhibitor), the trp-cage-crowder interactions are found to be specific, involving a few key residues, most of which are prolines. The effects of these crowders are contrasted with those of hard-sphere crowders. C 2015 AIP Publishing LLC. Equilibrium simulation of trp-cage in the presence of protein crowders[http://dx

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“…23 The peptide is simulated in homogeneous crowding environments, where either bovine pancreatic trypsin inhibitor (BPTI) or the B1 domain of streptococcal protein G (GB1) serves as crowding agent. Both these proteins are thermally highly stable 24,25 and therefore modeled using a fixed-backbone approximation, whereas the GB1m3 peptide is free to fold and unfold in the simulations. The simulations are conducted using MC dynamics at constant temperature.…”

Section: Introductionmentioning

confidence: 99%

Markov modeling of peptide folding in the presence of protein crowders

Nilsson

Mohanty

Irbäck

2018

The Journal of Chemical Physics

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We use Markov state models (MSMs) to analyze the dynamics of a β-hairpin-forming peptide in Monte Carlo (MC) simulations with interacting protein crowders, for two different types of crowder proteins [bovine pancreatic trypsin inhibitor (BPTI) and GB1]. In these systems, at the temperature used, the peptide can be folded or unfolded and bound or unbound to crowder molecules. Four or five major freeenergy minima can be identified. To estimate the dominant MC relaxation times of the peptide, we build MSMs using a range of different time resolutions or lag times. We show that stable relaxation-time estimates can be obtained from the MSM eigenfunctions through fits to autocorrelation data. The eigenfunctions remain sufficiently accurate to permit stable relaxation-time estimation down to small lag times, at which point simple estimates based on the corresponding eigenvalues have large systematic uncertainties. The presence of the crowders have a stabilizing effect on the peptide, especially with BPTI crowders, which can be attributed to a reduced unfolding rate k u , while the folding rate k f is left largely unchanged.

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Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters

Cited by 80 publications

References 36 publications

Partial characterization of a 36-kDa protein from Clostridium botulinum type E that inhibits trypsin and chymotrypsin.

Partial characterization of a 36-kDa protein from Clostridium botulinum type E that inhibits trypsin and chymotrypsin.

Equilibrium simulation of trp-cage in the presence of protein crowders

Markov modeling of peptide folding in the presence of protein crowders

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