Pracownie badawcze

Prof. dr hab. Jarosław Poznański

Pracownia Molekularnych Podstaw Aktywności Biologicznej

Zakres badań

We are combining computational and experimental methods to explain biochemical, biological, and medical observations. We currently focus on proteins, including their structure, stability, function, and interactions with substrates, small-molecule ligands, and other molecular partners. We also design and synthesize new molecular probes to study the thermodynamic contribution of particular types of interactions in model molecular systems.


Najważniejsze osiągnięcia badawcze

  • In collaboration with the Laboratory of Mass Spectrometry, we proposed the mechanism of Aβ1-40 peptide aggregation. The most important finding was that the experimentally determined fractal dimension of aggregates was 2, indicating the formation of flat sheet-like structures.
  • We explained molecular bases of the substrate preference of bacterial AlkB dioxygenase, which were further validated for human homologs (ALKBH3 and fat mass and obesity‐associated protein [FTO]).
  • Our long-term thermodynamics studies demonstrated that the contribution of halogen bonding to the free energy of ligand binding is predominated by a balance of electrostatic and hydrophobic interactions.

Opis badań

The main objective of our work is the development of research tools that enable building the relationship between molecular structure and biological function. Our research is based primarily on medium- and high-resolution models that are built on the basis of structural data that are available for similar systems (e.g., modeling by homology using Protein Data Bank structures as templates), in the context of biophysical observations (thermodynamic parameters, spectroscopic data, and HD exchange monitored by mass spectrometry [MS]), biochemical observations (kinetic data and substrate selectivity), biological observations (cell viability and drug metabolic pathways), and medical observations (molecular backgrounds of genetic diseases). Our goal is to identify specific interactions or combinations thereof that explain the observed effect. We sometimes act like passive researchers who only interpret observations (mainly in the case of medical data). For most problems, however, we are building hypotheses that provide feedback for further experimental research.

  • New algorithms of data analysis

We are constantly developing methodologies for the global analysis of data that are obtained under different conditions or even from different kinds of experimental approaches. We use state-of-art numerical models that allow the reliable estimation of parameters that are unavailable with standard software. We performed the first global analysis of MST (Microscale Thermophoresis) data for two independent binding sites,1 and interpreted the ITC data (Isothermal Titration Calorimetry) for the presence of three nonequivalent binding sites (doi: 10.1371/journal.pone.0154822). Some of our models were published in the highest ranked journals (doi: 10.1021/ja907567r, doi: 10.1038/s41467-019-11900-8).

  • Molecular modeling

Valuable molecular models that explain particular biophysical (e.g., the organization of peptide Aβ1-40 early stage aggregates, biochemical (e.g., substrate specificity of Arabidopsis DXO1 protein; doi: 10.1093/nar/gkz100), biological (doi: 10.1098/rsob.150238, doi: 10.3390/ijms21155268), or medical (doi: 10.15252/emmm.201809561, doi: 1002/ajmg.a.36646) observations have been proposed.

  • Halogen bonding and its thermodynamic contribution to the free energy of protein-ligand interactions

We are analyzing thermodynamic effects of individual halogen atoms on the interaction between halogenated ligands and their molecular target, the catalytic subunit of protein kinase CK2.1-4 We designed and synthesized dozens of molecular probes that contain various combinations of halogen atoms (F, Cl, Br, I) and studied their interactions with the molecular target using various thermodynamics techniques.5, 6 We synthesized a CK2 bisubstrate inhibitor with moderate activity. We also studied the geometry and topology of halogen bonds in protein-ligand complexes 7 and competition between halogen and hydrogen bonds.8

  • Hydrophobic interactions

A new hydrophobicity scale that is based on changes in solvent density that is caused by the solute has been proposed, enabling experimental determination of the apparent volume of a single molecule. We proved that the partial molar volume can be treated as a thermodynamic parameter that describes hydrophobicity. Each hydrophobic molecule affects the organization of the proximal solvent, and higher hydrophobicity of the solute is associated with a lower density of water in the solvation shell. We use this parameter to assess the hydrophobic contribution to the free energy of ligand binding.

  • Role of the Ada operon in the repair of exocyclic DNA lesions

We study mutagenic properties of several exocyclic DNA lesions, acrolein and chloroacetaldehyde adducts to cytosine and adenine, 1,N6-α-hydroxypropanoadenine,9 3,N4-α-hydroxypropanocytosine, 3,N4-α-hydroxyethano-cytosine, 3,N4-ethenocytosine (εC),10 and 1,N6-ethenoadenine (εA) and their repair by AlkB dioxygenase and AlkA glycosylase. AlkB acts according to a previously unknown DNA repair mechanism. It removes modifications from alkylated DNA bases via oxidative dealkylation, without disturbing DNA We experimentally proved that AlkB preferentially recognizes and repairs cationic substrates. We now focuse on the biological function of AidB dehydrogenase, characterizing DNA-AlkB-AidB, RNA-AlkB-AidB, and RNA-AlkB complexes, and studies the repair of exocyclic RNA adducts. This multidisciplinary approach should provide deeper insights into fundamental aspects of cellular homeostasis maintenance (NCN grant no. 2018/29/B/NZ3/02285).

  • Bibliography
  1. Winiewska et al. 2017. PLoS One 12.
  2. Wasiket et al. 2012. Journal of Physical Chemistry B 116, 7259-7268.
  3. Winiewska et al. 2015. Biochemical and Biophysical Research Communications 456, 282-287.
  4. Winiewska et al. 2015. Biochimica et Biophysica Acta-Proteins and Proteomics 1854, 1708-1717.
  5. Kasperowicz et al. 2020. Iubmb Life 72, 1211-1219.
  6. Marzec et al. 2020. Iubmb Life 72, 1203-1210.
  7. Poznanski et al. 2016. Acta Biochim. 63, 203-214.
  8. Poznanski et al. 2014. PLoS One 9.
  9. Dylewska et al. 2017. Biochem. J. 474, 1837-1852.
  10. Maciejewska et al. 2010 Mutat. Res. 684, 24-34.


  • In silico studies on proteins, their complexes, and modifications are based on structural models that are obtained via homology with the Yasara-Structure package. If desired, the standard force field can be extended based on ab initio The effects of residue replacements or modifications on protein stability or affinity for molecular targets are evaluated with the local UI for FoldX and AutoDock programs.
  • Thermodynamics studies on protein. Ligand interactions are performed with the aid of numerous biophysical methods. They include nanoDSF (equivalent to the thermal shift assay), microscale thermophoresis, and isothermal titration calorimetry. These data can be supplemented by affinity chromatography coupled with mass spectrometry and various nuclear magnetic resonance (NMR) techniques (13C, 15N, and 19F nuclear relaxation, relaxation-filtered NMR, diffusion ordered spectroscopy, and chemical shift perturbation).
  • Structural data can also be obtained by heteronuclear NMR or crystallography. We also support our studies with massive CSD and Protein Data Bank structural data screening.
  • Density data that are determined with Anton PAAR DMA 5000M enables the determination of partial molar volumes and thermal volumetric expansion at submilimolar concentrations. We use these data to study the contribution of hydrophobic solvation in biomolecular systems.
  • We collect biochemical data on “enzymes in action” with EU-OPENSCREEN facilities, but the assays for protein kinase and dioxygenase assays have already been implemented in our l
  • Our chemical laboratory is sufficiently equipped to perform various types of chemical syntheses, including halogenation reactions and reactions in anhydrous conditions over a wide temperature range (-70°C to over 200°C). This allows the multi-step synthesis of designed heterogeneously halogenated heterocyclic compounds or their libraries. We are able to perform multi-component reactions and synthesize substrates for these reactions (e.g., isocyanates). We are able to synthesize bisubstrate ligands of target proteins using a multicomponent reaction.


Publikacje (z afiliacją IBB PAN)

KACZKA P., WINIEWSKA M., ZHUKOV I., REMPOŁA B., BOLEWSKA K., ŁOZIŃSKI T., EJCHART A., POZNAŃSKA A., WIERZCHOWSKI K.L., POZNAŃSKI J., The TFE-induced transient native-like structure of the intrinsically disordered σ704 domain of Escherichia coli RNA polymerase. European Biophysics Journal (2014) 43(12): 581-594 IF 2.474
CZERWIŃSKA J., POZNAŃSKI J., DĘBSKI J., BUKOWY Z., BOHR V.A., TUDEK B., SPEINA E., Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal. Nucleic Acids Research (2014) 42(17): 11119-11135 IF 8.808
GOS M., FAHIMINIYA S., POZNAŃSKI J., KLAPECKI J., OBERSZTYN E., PIOTROWICZ M., WIERZBA J., POSMYK R., BAL J., MAJEWSKI J., Contribution of RIT1 mutations to the pathogenesis of noonan syndrome: four new cases and further evidence of heterogeneity. American Journal of Medical Genetics-PART A (2014) 164A(9): 2310-2316 IF 2.048
SITKIEWICZ E., KŁONIECKI M., POZNAŃSKI J., BAL W., DADLEZ M., Factors influencing compact-extended structure equilibrium in oligomers of Aβ1-40 peptide-an ion mobility mass spectrometry study. Journal of Molecular Biology (2014) 426(15): 2871-2885 IF 3.959
HOFFMAN-ZACHARSKA D., MIERZEWSKA H., SZCZEPANIK E., POZNAŃSKI J., MAZURCZAK T., JAKUBIK-TOMASZUK A., MĄDRY J., KIERDASZUK A., BAL J., The spectrum of PLP1 gene mutations in patients with the classical form of the Pelizaeus-Merzbacher disease. Developmental Period Medicine/ Medycyna Wieku Rozwojowego (2013) XVII (4): 293-300 IF-
HOFFMAN-ZACHARSKA D., KOZIOROWSKI D., ROSS O.A., MILEWSKI M., POZNAŃSKI J., JUREK M.,WSZOŁEK Z.K., SOTO-ORTOLAZA A., SŁAWEK J., JANIK P., JAMROZIK Z., POTULSKA-CHROMIK A., JASIŃSKA-MYGA B., OPALA G., KRYGOWSKA-WAJS A., CZYŻEWSKI K., DICKSON D.W., BAL J., FRIEDMAN A., Novel A18T and pA29S substitutions in α-synuclein may be associated with sporadic Parkinson's disease. Parkinsonism & Related Disorders (2013) 19: 1057-1060 IF 3.274
ZAWISZA I., RÓZGA M., POZNAŃSKI J., BAL W., Cu(II) complex formation by ACES buffer. Journal of Inorganic Biochemistry (2013) 129: 58-61 IF 3.197
HOFFMAN-ZACHARSKA D., KMIEĆ T., POZNAŃSKI J., JUREK M.,BAL J., Mutations in the PLP1 gene residue p. Gly198 as the molecular basis of Pelizeaus-Merzbacher phenotype. Brain & Development (2013) 35(9): 877-880 IF 1,668
MIELECKI D., SAUMAA S., WRZESIŃSKI M., MACIEJEWSKA A.M., ŻUCHNIEWICZ K., SIKORA A., PIWOWARSKI J., NIEMINUSZCZY J., KIVISAAR M., GRZESIUK E., Pseudomonas putida AlkA and AlkB proteins comprise different defense systems for the repair of alkylation damage to DNA – In Vivo, In Vitro, and In Silico studies. PLOS ONE (2013) 8(10): e76198 (24 p.) IF 3.730
PROTAS A.M., ARIANI H.H.N., BONNA A., POLKOWSKA-NOWAKOWSKA A., POZNAŃSKI J., BAL W., Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering: Active sequence optimization. Journal of Inorganic Biochemistry (2013) 127: 99-106 IF 3.197
POZNAŃSKI J., SHUGAR D., Halogen bonding at the ATP binding site of protein kinases: Preferred geometry and topology of ligand binding. Biochimica et Biophysica Acta - Proteins and Proteomics (2013) 1834(7): 1381-1386 IF 3.733
CHARZEWSKA A., NAWARA M., JAKUBIUK-TOMASZUK A., OBERSZTYN E., HOFFMAN-ZACHARSKA D., ELERT E., JUREK M.,BARTNIK M., POZNAŃSKI J., BAL J., Expanding the phenotype associated with missense mutations of the ARX gene. American Journal of Medical Genetics Part A (2013) 161A: 1813-1816 IF 2.304
POZNAŃSKI J., SZCZĘSNY P., RUSZCZYŃSKA K., ZIELENKIEWICZ P., PĄCZEK L., Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm. Biochemical and Biophysical Research Communications (2013) 430(2): 741-744 IF 2.406
MACIEJEWSKA A.M., POZNAŃSKI J., KACZMARSKA Z., KROWISZ B., NIEMINUSZCZY J., POLKOWSKA-NOWAKOWSKA A., GRZESIUK E., KUŚMIEREK J.T., AlkB dioxygenase preferentially repairs protonated substrates: specificity against exocyclic adducts and molecular mechanism of action. Journal of Biological Chemistry (2013) 288(1): 432-441 IF 4.651
LENARČIČ-ŽIVKOVIĆ M., ZARĘBA-KOZIOŁ M., ZHUKOVA L., POZNAŃSKI J., ZHUKOV I., WYSŁOUCH-CIESZYŃSKA A., Post-translational S-nitrosylation is an endogenous factor fine tuning the properties of human S100A1 protein. Journal of Biological Chemistry (2012) 287(48): 40457-40470 IF 4.773
WĄSIK R., WIŃSKA P., POZNAŃSKI J., SHUGAR D., Isomeric mono-, di-, and tri-bromobenzo-1H-triazoles as inhibitors of human protein kinase CK2α. PLOS ONE (2012) 7(11): e48898 IF 4.092