Phage Display Selection of P1 Mutants of BPTI Directed against Five Different Serine Proteinases

1999 ◽  
Vol 380 (1) ◽  
Author(s):  
L. Kiczak ◽  
K. Koscielska ◽  
J. Otlewski ◽  
M. Czerwinski ◽  
M. Dadlez
Keyword(s):  
Phage Display ◽  
Trypsin Inhibitor ◽  
Association Constant ◽  
Serine Proteinases ◽  
Wild Type ◽  
Association Energy ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor ◽  
M13 Phage ◽  
Selection Of

AbstractThe P1 position of protein inhibitors and oligopeptide substrates determines, to a large extent, association energy with many serine proteinases. To test the agreement of phage display selection with the existing thermodynamic data, a small library of all 20 P1 mutants of basic pancreatic trypsin inhibitor (BPTI) was created, fused to protein III, and displayed on the surface of M13 phage. The wild type of displayed inhibitor monovalently and strongly inhibited trypsin with an association constant of

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Lys77-plasmin

1986 ◽  
Vol 191 (2) ◽  
pp. 295-297 ◽  
Author(s):  
Enea Menegatti ◽  
Mario Guarneri ◽  
Martino Bolognesi ◽  
Paolo Ascenzi ◽  
Gino Amiconi
Keyword(s):  
Trypsin Inhibitor ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor

scholarly journals Selection of Peptides Targeting Helix 31 of Bacterial 16S Ribosomal RNA by Screening M13 Phage-Display Libraries

Molecules ◽  
2011 ◽  
Vol 16 (2) ◽  
pp. 1211-1239 ◽  
Author(s):  
Tek N. Lamichhane ◽  
N. Dinuka Abeydeera ◽  
Anne-Cécile E. Duc ◽  
Philip R. Cunningham ◽  
Christine S. Chow
Keyword(s):  
Phage Display ◽  
Ribosomal Rna ◽  
16S Ribosomal Rna ◽  
Phage Display Libraries ◽  
M13 Phage ◽  
Selection Of

scholarly journals Free Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitor Using Metadynamics

2021 ◽  
Author(s):  
Pär Söderhjelm ◽  
Mandar Kulkarni
Keyword(s):  
Free Energy ◽  
Trypsin Inhibitor ◽  
Side Chain ◽  
Side Chains ◽  
Aromatic Residues ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor ◽  
Energy Surfaces ◽  
Aromatic Side Chains

Aromatic side-chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cβ-Cγ axis (χ2 dihedral of side-chain) producing two symmetry-equivalent states. The ring-flip dynamics act as an NMR probe to understand local conformational fluctuations. Ring-flips are categorized as slow (ms onwards) or fast (ns to near ms) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to discriminate between slow and fast ring-flips for eight individual aromatic side-chains (F4, Y10, Y21, F22, Y23, F33, Y35, F45) of basic pancreatic trypsin inhibitor (BPTI). Well-tempered metadynamics simulations were performed to observe ring-flipping free energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to classify fast and slow ring-flips. Most of the residues needed χ1 (N−Cχα) as a complementary CV, indicating the importance of librational motions in ring-flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events are observed for residues F22 and F33, indicating a possible role of friction effects in the ring-flipping. The results demonstrate the successful application of the metadynamics based approach to estimate ring-flip rates of aromatic residues in BPTI and identify certain limitations of the approach.

scholarly journals Transgenic expression of pancreatic secretory trypsin inhibitor-1 rescues SPINK3-deficient mice and restores a normal pancreatic phenotype

2010 ◽  
Vol 298 (4) ◽  
pp. G518-G524 ◽  
Author(s):  
Joelle M.-J. Romac ◽  
Masaki Ohmuraya ◽  
Cathy Bittner ◽  
M. Faraz Majeed ◽  
Steven R. Vigna ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Survival Rates ◽  
Acinar Cells ◽  
Threshold Level ◽  
Protective Role ◽  
Trypsin Inhibitors ◽  
Pancreatic Acinar Cells ◽  
Wild Type ◽  
Transgenic Expression ◽  
Pancreatic Trypsin Inhibitor

Endogenous trypsin inhibitors are synthesized, stored, and secreted by pancreatic acinar cells. It is believed that they play a protective role in the pancreas by inhibiting trypsin within the cell should trypsinogen become prematurely activated. Rodent trypsin inhibitors are highly homologous to human serine protease inhibitor Kazal-type 1 (SPINK1). The mouse has one pancreatic trypsin inhibitor known as SPINK3, and the rat has two trypsin inhibitors commonly known as pancreatic secretory trypsin inhibitors I and II (PSTI-I and -II). Rat PSTI-I is a 61-amino acid protein that shares 65% sequence identity with mouse SPINK3. It was recently demonstrated that mice with genetic deletion of the Spink3 gene ( Spink3−/− ) do not survive beyond 15 days and lack normal pancreata because of pancreatic autophagy. We have shown that targeted transgenic expression of the rat Psti1 gene to acinar cells in mice [ TgN(Psti1)] protects mice against caerulein-induced pancreatitis. To determine whether the autophagic phenotype and lethality in Spink3−/− mice were due to lack of pancreatic trypsin inhibitor, we conducted breeding studies with Spink3+/− heterozygous mice and TgN(Psti1) mice. We observed that, whereas Spink3+/+, Spink3+/−, and Spink3−/− /TgN(Psti1) mice had similar survival rates, no Spink3−/− mice survived longer than 1 wk. The level of expression of SPINK3 protein in acini was reduced in heterozygote mice compared with wild-type mice. Furthermore, endogenous trypsin inhibitor capacity was reduced in the pancreas of heterozygote mice compared with wild-type or knockout mice rescued with the rat Psti1 gene. Surprisingly, the lesser amount of SPINK3 present in the pancreata of heterozygote mice did not predispose animals to increased susceptibility to caerulein-induced acute pancreatitis. We propose that a threshold level of expression is sufficient to protect against pancreatitis.

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