Search results

1

A novel, stable, helical scaffold as an alternative binder - construction of phage display libraries

100%

Cyranka-Czaja A., Otlewski J.

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2012

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vol. 59

|

issue 3

383-390

EN

Specific, high affinity binding macromolecules are of great importance for biomedical and biotechnological applications. The most popular classical antibody-based molecules have recently been challenged by alternative scaffolds with desirable biophysical properties. Phage display technology applied to such scaffolds allows generation of potent affinity reagents by in vitro selection. Here, we report identification and characterization of a novel helical polypeptide with advantageous biophysical properties as a template for construction of phage display libraries. A three-helix bundle structure, based on Measles virus phosphoprotein P shows a very favourable stability and solubility profile. We designed, constructed and characterized six different types of phage display libraries based on the proposed template. Their functional size of over 109 independent clones, balanced codon bias and decent display level are key parameters attesting to the quality and utility of the libraries. The new libraries are a promising tool for isolation of high affinity binders based on a small helical scaffold which could become a convenient alternative to antibodies.

2

The serine proteinase inhibitor from summer squash (Cucurbita pepo): some structural features, stability and proteolytic degradation

100%

Otlewski J., Wilusz T.

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vol. 32

|

issue 4

285-293

3

PDZ domain from Dishevelled - a specificity study

81%

Śmietana K., Mateja A., Krężel A., Otlewski J.

|

2011

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vol. 58

|

issue 2

243-250

EN

Intracellular signaling cascades induced by Wnt proteins play a key role in developmental processes and are implicated in cancerogenesis. It is still unclear how the cell determines which of the three possible Wnt response mechanisms should be activated, but the decision process is most likely dependent on Dishevelled proteins. Dishevelled family members interact with many diverse targets, however, molecular mechanisms underlying these binding events have not been comprehensively described so far. Here, we investigated the specificity of the PDZ domain from human Dishevelled-2 using C-terminal phage display, which led us to identification of a leucine-rich binding motif strongly resembling the consensus sequence of a nuclear export signal. PDZ interactions with several peptide and protein motifs (including the nuclear export signal sequence from Dishevelled-2 protein) were investigated in detail using fluorescence spectroscopy, mutational analysis and immunoenzymatic assays. The experiments showed that the PDZ domain can bind the nuclear export signal sequence of the Dishevelled-2 protein. Since the intracellular localization of Dishevelled is governed by nuclear localization and nuclear export signal sequences, it is possible that the intramolecular interaction between PDZ domain and the export signal could modulate the balance between nuclear and cytoplasmic pool of the Dishevelled protein. Such a regulatory mechanism would be of utmost importance for the differential activation of Wnt signaling cascades, leading to selective promotion of the nucleus-dependent Wnt β-catenin pathway at the expense of non-canonical Wnt signaling.

4

PDZ domains - common players in the cell signaling.

81%

Jeleń F., Oleksy A., Śmietana K., Otlewski J.

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2003

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vol. 50

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issue 4

985-1017

EN

PDZ domains are ubiquitous protein interaction modules that play a key role in cellular signaling. Their binding specificity involves recognition of the carboxyl-terminus of various proteins, often belonging to receptor and ion channel families. PDZ domains also mediate more complicated molecular networks through PDZ-PDZ interactions, recognition of internal protein sequences or phosphatidylinositol moieties. The domains often form a tandem of multiple copies, but equally often such tandems or single PDZ domain occur in combination with other signaling domains (for example SH3, DH/PH, GUK, LIM, CaMK). Common occurrence of PDZ domains in Metazoans strongly suggests that their evolutionary appearance results from the complication of signaling mechanisms in multicellular organisms. Here, we focus on their structure, specificity and role in signaling pathways.

5

Structure of small G proteins and their regulators.

81%

Paduch M., Jeleń F., Otlewski J.

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2001

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vol. 48

|

issue 4

829-850

EN

In recent years small G proteins have become an intensively studied group of regulatory GTP hydrolases involved in cell signaling. More than 100 small G proteins have been identified in eucaryotes from protozoan to human. The small G protein superfamily includes Ras, Rho Rab, Rac, Sar1/Arf and Ran homologs, which take part in numerous and diverse cellular processes, such as gene expression, cytoskeleton reorganization, microtubule organization, and vesicular and nuclear transport. These proteins share a common structural core, described as the G domain, and significant sequence similarity. In this paper we review the available data on G domain structure, together with a detailed analysis of the mechanism of action. We also present small G protein regulators: GTPase activating proteins that bind to a catalytic G domain and increase its low intrinsic hydrolase activity, GTPase dissociation inhibitors that stabilize the GDP-bound, inactive state of G proteins, and guanine nucleotide exchange factors that accelerate nucleotide exchange in response to cellular signals. Additionally, in this paper we describe some aspects of small G protein interactions with downstream effectors.

6

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

81%

Petters E., Krowarsch D., Otlewski J.

|

2013

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vol. 60

|

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.

7

Novel peptide recognized by RhoA GTPase

81%

Drulis-Fajdasz D., Jelen F., Oleksy A., Otlewski J.

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2006

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vol. 53

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issue 3

515-524

EN

A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA - R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+- and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.

8

Trypsin inhibitors in summer squash (Cucurbita pepo) seeds. Isolation, purification and partial characterization of the inhibitors

81%

Otlewski J., Antoni Polanowski J. L., Wilusz T.

Acta Biochimica Polonica

|

1984

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vol. 31

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issue 3

267-278

9

Preparation and characteristics of trypsin inhibitors from the seeds of squash (Cucurbita maxima) and zucchini (Cucurbita pepo, vcir. Giromontia)

71%

Leluk J., Otlewski J., Wieczorek M., Polanowski A., Wilusz T.

Acta Biochimica Polonica

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1983

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vol. 30

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issue 2

127-138

10

Degenerate specificity of PDZ domains from RhoA-specific nucleotide exchange factors PDZRhoGEF and LARG

62%

Smietana K., Kasztura M., Paduch M., Derewenda U., Derewenda Z., Otlewski J.

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2008

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vol. 55

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issue 2

269-280

EN

PDZ domains are ubiquitous protein-protein interaction modules which bind short, usually carboxyterminal fragments of receptors, other integral or membrane-associated proteins, and occasionally cytosolic proteins. Their role in organizing multiprotein complexes at the cellular membrane is crucial for many signaling pathways, but the rules defining their binding specificity are still poorly understood and do not readily explain the observed diversity of their known binding partners. Two homologous RhoA-specific, multidomain nucleotide exchange factors PDZRhoGEF and LARG contain PDZ domains which show a particularly broad recognition profile, as suggested by the identification of five diverse biological targets. To investigate the molecular roots of this phenomenon, we constructed a phage display library of random carboxyterminal hexapeptides. Peptide variants corresponding to the sequences identified in library selection were synthesized and their affinities for both PDZ domains were measured and compared with those of peptides derived from sequences of natural partners. Based on the analysis of the binding sequences identified for PDZRhoGEF, we propose a sequence for an 'optimal' binding partner. Our results support the hypothesis that PDZ-peptide interactions may be best understood when one considers the sum of entropic and dynamic effects for each peptide as a whole entity, rather than preferences for specific residues at a given position.

11

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants

62%

Polanowski A., Cieślar E., Otlewski J., Nienartowicz B., Wilimowska-Pelc A., Wilusz T.

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issue 4

395-406

12

Phage display of proteins

61%

Kościelska K., Kiczak L., Kasztura M., Wesołowska O., Otlewski J.

Acta Biochimica Polonica

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1998

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vol. 45

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issue 3

705-720

13

Structural and energetic aspects of protein-protein recognition

60%

Otlewski J., Apostoluk W.

Acta Biochimica Polonica

|

1997

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vol. 44

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issue 3

367-387

14

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

52%

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

|

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.

15

Serine proteinase from Cucurbita ficifolia seeds

50%

Dryjański M., Otlewski J., Wilusz T.

Acta Biochimica Polonica

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1990

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vol. 37

|

issue 1

169-172

16

Protein inhibitors of serine proteinases

50%

Otlewski J., Krowarsch D., Apostoluk W.

Acta Biochimica Polonica

|

1999

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vol. 46

|

issue 3

531-565

17

Squash inhibitor family of serine proteinases

50%

Otlewski J., Krowarsch D.

Acta Biochimica Polonica

|

1996

|

vol. 43

|

issue 3

431-444

Refine search results

A novel, stable, helical scaffold as an alternative binder - construction of phage display libraries

The serine proteinase inhibitor from summer squash (Cucurbita pepo): some structural features, stability and proteolytic degradation

PDZ domain from Dishevelled - a specificity study

PDZ domains - common players in the cell signaling.

Structure of small G proteins and their regulators.

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

Novel peptide recognized by RhoA GTPase

Trypsin inhibitors in summer squash (Cucurbita pepo) seeds. Isolation, purification and partial characterization of the inhibitors

Preparation and characteristics of trypsin inhibitors from the seeds of squash (Cucurbita maxima) and zucchini (Cucurbita pepo, vcir. Giromontia)

Degenerate specificity of PDZ domains from RhoA-specific nucleotide exchange factors PDZRhoGEF and LARG

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants

Phage display of proteins

Structural and energetic aspects of protein-protein recognition

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

Serine proteinase from Cucurbita ficifolia seeds

Protein inhibitors of serine proteinases

Squash inhibitor family of serine proteinases