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1

Design, expression and characterization of a highly stable tetratricopeptide-based protein scaffold for phage display application

100%

Petters E., Krowarsch D., Otlewski J.

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2013

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vol. 60

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issue 4

585-590

EN

Tetratricopeptide repeat (TPR) is a structural motif mediating variety of protein-protein interactions. It has a high potential to serve as a small, stable and robust, non-immunoglobulin ligand binding scaffold. In this study, we showed the consensus approach to design the novel protein called designed tetratricopeptide repeat (dTPR), composed of three repeated 34 amino-acid tetratricopeptide motifs. The designed sequence was efficiently overexpressed in E. coli and purified to homogeneity. Recombinant dTPR is monomeric in solution and preserves its secondary structure within the pH range from 2.0 to 11.0. Its denaturation temperature at pH 7.5 is extremely high (104.5°C) as determined by differential scanning calorimetry. At extreme pH values the protein is still very stable: denaturation temperature is 90.1°C at pH 2.0 and 60.4°C at pH 11. Chemical unfolding of the dTPR is a cooperative, two-state process both at pH 7.5 and 2.0. The free energy of denaturation in the absence of denaturant equals to 15.0 kcal/mol and 13.5 kcal/mol at pH 7.5 and 2.0, respectively. Efficient expression and extraordinary biophysical properties make dTPR a promising framework for a biotechnological application, such as generation of specific ligand- binding molecules.

2

Structure-function relationship of serine protease-protein inhibitor interaction.

64%

Otlewski J., Jaskólski M., Buczek O., Cierpicki T., Czapińska H., Krowarsch D., Smalas A. O., Stachowiak D., Szpineta A., Dadlez M.

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2001

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vol. 48

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issue 2

419-428

EN

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calorimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.

3

Protein inhibitors of serine proteinases

63%

Otlewski J., Krowarsch D., Apostoluk W.

Acta Biochimica Polonica

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1999

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vol. 46

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issue 3

531-565

4

Squash inhibitor family of serine proteinases

62%

Otlewski J., Krowarsch D.

Acta Biochimica Polonica

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1996

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vol. 43

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issue 3

431-444

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4 Acta Biochimica Polonica

1 2013

1 2001

1 1999

1 1996

4 Krowarsch D.

4 Otlewski J.

1 Apostoluk W.

1 Buczek O.

1 Cierpicki T.

1 Czapińska H.

1 Dadlez M.

1 Jaskólski M.

1 Petters E.

1 Smalas A. O.

1 Stachowiak D.

1 Szpineta A.

Design, expression and characterization of a highly stable tetratricopeptide-based protein scaffold for phage display application

Structure-function relationship of serine protease-protein inhibitor interaction.

Protein inhibitors of serine proteinases

Squash inhibitor family of serine proteinases