Wyniki wyszukiwania

1

Inżynieria białka. Sekcja 4 Pierwszego Krajowego Kongresu Biotechnologii

100%

Otlewski J.

Biotechnologia

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2000

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nr 1

2

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

63%

Otlewski J., Wilusz T.

Acta Biochimica Polonica

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1985

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tom 32

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nr 4

3

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

63%

Cyranka-Czaja A., Otlewski J.

Acta Biochimica Polonica

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2012

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tom 59

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nr 3

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.

4

Squash inhibitor family of serine proteinases

63%

Otlewski J., Krowarsch D.

Acta Biochimica Polonica

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1996

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tom 43

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nr 3

Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (Pl-Pl') is between residue 5 (Lys, Arg or Leu) and 6 (always lie). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-strai:ded antiparallel p-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carbo- xypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor P2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: Xa and XII*, cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exibit at neutral pH a high fccWKm index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant.

5

Structural and energetic aspects of protein-protein recognition

63%

Otlewski J., Apostoluk W.

Acta Biochimica Polonica

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1997

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tom 44

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nr 3

Specific recognition between proteins plays a crucial role in a great number of vital processes. In this review different types of protein-protein complexes are analyzed on the basis of their three-dimensional structures which became available in recent years. The complexes which are analyzed include: those resulting from different types of recognition between proteinase and protein inhibitor (canonical inhibitors of serine proteinases, hirudin, inhibitors of cysteine proteinases, carboxypeptidase inhibitor), barnase-barstar, human growth hormone-receptor and antibody-antigen. It seems obvious that specific and strong protein-protein recognition is achieved in many different ways. To further explore this question, the structural information was analyzed together with kinetic and thermodynamic data available for the respective complexes. It appears that the energy and rates of specific recognition of proteins are influenced by many different factors, including: area of interacting surfaces; complementarity of shapes, charges and hydrogen bonds; water structure at the interface; conformational changes; additivity and cooperativity of individual interactions, steric effects and various (conformational, hydration) entropy changes.

6

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

51%

Petters E., Krowarsch D., Otlewski J.

Acta Biochimica Polonica

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2013

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tom 60

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nr 4

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

51%

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

Acta Biochimica Polonica

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2006

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tom 53

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nr 3

8

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

51%

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

Acta Biochimica Polonica

|

1984

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tom 31

|

nr 3

9

PDZ domain from Dishevelled - a specificity study

51%

Smietana K., Mateja A., Krezel A., Otlewski J.

Acta Biochimica Polonica

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2011

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tom 58

|

nr 2

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.

10

Serine proteinase from Cucurbita ficifolia seeds

51%

Dryjanski M., Otlewski J., Wilusz D.

Acta Biochimica Polonica

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1990

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tom 37

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nr 1

11

Structure of small G proteins and their regulators

51%

Paduch M., Jelen F., Otlewski J.

Acta Biochimica Polonica

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2001

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tom 48

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nr 4

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.

12

Protein inhibitors of serine proteinases

51%

Otlewski J., Krowarsch D., Apostoluk W.

Acta Biochimica Polonica

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1999

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tom 46

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nr 3

Serine proteinases and their natural protein inhibitors belong to the most intensively studied models of protein-protein recognition. Protein inhibitors do not form a single group but can be divided into about 20 different families. Global structures of proteins representing different inhibitor families are completely different and comprise α-helical proteins, β-sheet proteins,α/β-proteins and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished: canonical (standard mechanism) inhibitors, non-canonical inhibitors, and serpins. The canonical inhibitor binds to the enzyme through the exposed and convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is consistently very similar and resembles that of an ideal substrate. Non-canonical inhibitors, originating from blood sucking organisms, specifically block enzymes of the blood clotting cascade. The interaction is mediated through inhibitor N-terminus which binds to the proteinase forming a parallel β-sheet. There are also extensive secondary interactions which provide an additional buried area and contribute significantly to the strength and specificity of recognition. Serpins are major proteinase inhibitors occurring in plasma. Similarly to canonical inhibitors, serpins interact with their target proteinases in a substrate-like manner. However, in the case of serpins, cleavage of a single peptide bond in a flexible and exposed binding loop leads to dramatic structural changes.

13

PDZ domains - common players in the cell signalling

51%

Jelen F., Oleksy A., Smietana K., Otlewski J.

Acta Biochimica Polonica

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2003

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tom 50

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nr 4

PDZ domains are ubiquitous protein interaction modules that play a key role in cellular signaling. Their binding specificity involves recognition of the carboxyl-termi- nus 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.

14

Profesor Michael Laskowski, Junior 1930-2004

50%

Otlewski J.

Postępy Biochemii

|

2004

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tom 50

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nr 4

3

15

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

45%

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

Acta Biochimica Polonica

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1983

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tom 30

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nr 2

16

Phage display of proteins

45%

Koscielska K., Kiczak L., Kasztura M., Wesolowska O., Otlewski J.

Acta Biochimica Polonica

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1998

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tom 45

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nr 3

In recent years the phage display approach has become an increasingly popular method in protein research. This method enables the presentation of large peptide and protein libraries on the surface of phage particles from which molecules of desired functional property(ies) can be rapidly selected. The great advantage of this method is a direct linkage between an observed phenotype and encapsulated genotype, which allows fast determination of selected sequences. The phage display approach is a powerful tool in generating highly potent biomolecules, including: search for specific antibodies, determining enzyme specificity, exploring protein-protein and protein-DNA interactions, minimizing proteins, introducing new functions into different protein scaffolds, and searching sequence space of protein folding. In this article many examples are given to illustrate that this technique can be used in different fields of protein science. The phage display has a potential of the natural evolution and its possibilities are far beyond rational prediction. Assuming that we can design the selection agents and conditions we should be able to engineer any desired protein function or feature.

17

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

39%

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

Acta Biochimica Polonica

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2008

|

tom 55

|

nr 2

269-280

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.

18

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants

39%

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

Acta Biochimica Polonica

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1987

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tom 34

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nr 4

19

Structural and thermodynamic aspects of plant-hormone binding by CSBP

33%

Pasternak O., Bujacz G. D., Yasuyuki F., Hashimoto Y., Sikorski M. M., Jaskolski M., Jelen F., Otlewski J.

Acta Biochimica Polonica

|

2006

|

tom 53

|

nr Supplement 1

20

Structure-function relationship of serine protease-protein inhibitor interaction

33%

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

Acta Biochimica Polonica

|

2001

|

tom 48

|

nr 2

We re port our prog ress in un der stand ing the struc ture-function re la tion ship of the interaction between protein inhibitors and several serine proteases. Recently, we have de ter mined high res o lu tion so lu tion struc tures of two in hib i tors Apis mellifera chymotrypsin in hib i tor-1 (AMCI-I) and Linum usitatissimumtrypsin in hib i tor (LUTI) in the free state and an ul tra high res o lu tion X-ray struc ture of BPTI. All three in hib i tors, de spite to tally dif fer ent scaf folds, con tain a sol vent ex posed loop of sim i lar con- for ma tion which is highly com ple men tary to the en zyme ac tive site. Iso ther mal calorim e try data show that the in ter ac tion be tween wild type BPTI and chymotrypsin is entropy driven and that the enthalpy com po nent op poses com plex for ma tion. Our research is fo cused on ex ten sive mu ta gen e sis of the four po si tions from the pro te ase bind ing loop of BPTI: P1, P1', P3, and P4. We mu tated these res i dues to dif fer ent amino ac ids and the vari ants were char ac ter ized by de ter mi na tion of the as so ci a tion con stants, sta bil ity pa ram e ters and crys tal struc tures of pro te ase–in hib i tor complexes. Ac com mo da tion of the P1 res i due in the S1 pocket of four pro teas es: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 vari ants. High res o lu tion X-ray struc tures of ten com plexes be tween bo vine trypsin and P1 vari ants of BPTI have been de ter mined and com pared with the cog nate P1 Lysside chain. Mu ta tions of the wild type Ala16 (P1') to larger side chains al ways caused a drop of the as so ci a tion con stant. Ac cord ing to the crys tal struc ture of the Leu16 BPTI–trypsin com plex, in tro duc tion of the larger res i due at the P1' po si tion leads to steric con flicts in the vi cin ity of the mu ta tion. Finally, mu ta tions at the P4 site al lowed an im prove ment of the as so ci a tion with sev eral serine pro teas es in volved in blood clot ting. Con versely, in tro duc tion of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing ef fect on the com plex with these pro teas es.

Ograniczanie wyników

Inżynieria białka. Sekcja 4 Pierwszego Krajowego Kongresu Biotechnologii

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

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

Squash inhibitor family of serine proteinases

Structural and energetic aspects of protein-protein recognition

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 three inhibitors

PDZ domain from Dishevelled - a specificity study

Serine proteinase from Cucurbita ficifolia seeds

Structure of small G proteins and their regulators

Protein inhibitors of serine proteinases

PDZ domains - common players in the cell signalling

Profesor Michael Laskowski, Junior 1930-2004

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

Phage display of proteins

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

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants

Structural and thermodynamic aspects of plant-hormone binding by CSBP

Structure-function relationship of serine protease-protein inhibitor interaction