PROJEKT ProtModStruct​

IP-2014-09-4274

Trajanje:

01.06.2015. – 31.05.2019.

Financiranje / Funding:
HRVATSKA ZAKLADA ZA ZNANOST / CROATIAN SCIENCE FOUNDATION

VODITELJ PROJEKTA / PRINCIPAL INVESTIGATOR: prof. dr. sc. Dubravka Matković-Čalogović

INSTITUCIJE KOJE SUDJELUJU U PROJEKTU / INSTITUTIONS

Prirodoslovno-matematički fakultet Sveučilište u Zagrebu, Zagreb, Hrvatska / Faculty of Science, University of Zagreb, Croatia

Institut za medicinska istraživanja i medicinu rada, Zagreb, Hrvatska / Institute for Medical Research and Occupational Health, Zagreb, Croatia

Department of Biomedical Sciences, University of Padua, Padua, Italy

Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Njemačka

National Institute of Chemistry, Slovenian NMR Centre, Ljubljana, Slovenia

NAZIV PROJEKTA / PROJECT TITLE

Esencijalni metalni ioni u proteinima iz Helicobacter pylori i modelnim spojevima - struktura i funkcija/svojstvo

  Essential metal ions in Helicobacter pylori proteins and model complexes – structure and       function/property

SAŽETAK

U okviru istraživanja na ovom projektu želimo strukturno karakterizirati nekoliko proteina iz Helicobacter pylori (i odrediti njihovu funkciju) kao i modelne komplekse malih molekula sa esencijalnim metalnim ionima i biološki važnim ligandima (i odrediti njihova svojstva). Prioritetni će biti metali koji se nalaze u metaloproteinima iz H. pylori. Svjetska zdravstvena organizacija proglasila je H. pylori karcinogenom 1. klase te je važna meta istraživanja zbog svoje patogenosti. Proteini HypA i HypB su važni za pravilnu konstrukciju metalnog centra [Ni,Fe] hidrogenaze i urease. Proteini CrdA i CrdB potrebni su za održavanje koncentracije slobodnih iona bakra u citoplazmi ispod toksične razine. Namjeravamo klonirati, eksprimirati, pročistiti i karakterizirati kompleks HypA-HypB te proteine CrdA i CrdB biofizičkim i strukturnim metodama (termalna stabilnost, cirkularni dikroizam, određivanje konstante disocijacije bakrovog iona, rentgenska difrakcija, 3D NMR). Želimo odrediti funkciju metalošaperona u unutarstaničnoj regulaciji nikla (HypA-HypB) kao i mehanizam regulacije koncentracije bakra (CrdA, CrdB).

Istraživat ćemo komplekse metala (bakra, nikla, željeza i drugih esencijalnih metala) s ciljem da odredimo preferirano koordinacijsko okruženje metala s aminokiselinama i derivatima aminokiselina (N-alkiliranih, amida, peptida, peptida bogatih metioninom i drugih) te dobijemo informaciju o specifičnosti aminokiselinskog ili peptidnog pokrajnjeg lanca prema različitim metalnim ionima. Komplekse ćemo sintetizirati te ih karakterizirati termičkim i spektroskopskim metodama i rentgenskom strukturnom analizom. Konstruirat ćemo konformacije bakrovih kompleksa s aminokiselinama te optimizirati njihovu geometriju pomoću kvantno-kemijskih proračuna da bismo istražili njihove moguće ravnotežne geometrije.

ABSTRACT

Within this project we will structurally characterize several proteins from Helicobacter pylori (and reveal their functions) and small molecule model complexes of essential metal ions with biologically relevant ligands (and find their properties). The priority will be the essential metals involved in H. pylori metalloproteins.The World Health Organization has declared the H. pylori a Class 1 carcinogen and it is an important target for research due to its pathological relevance. H. pylori requires several accessory proteins such as HypA and HypB for the proper construction of the [Ni,Fe] hydrogenase and urease. The resistance determinants CrdA and CrdB are required for keeping the concentration of free copper ions in the cytoplasm below toxic levels. Our aim is to clone, express, purify and characterize the complex HypA-HypB and proteins CrdA and CrdB by biophysical and structural methods (thermal stability assay, circular dichroism, determination of copper ion dissociation constant, X-ray diffraction, SAXS, 3D NMR). We propose to determine the role of the metal ions in these proteins, function of the metallochaperones in intracellular nickel delivery (HypA-HypB) and the mechanism of copper level regulation (CrdA, CrdB).

The aim of the study of the metal complexes (with copper, nickel, iron, zinc and other essential metal ions) is to determine how these metals template their coordination environment by amino acids and their derivatives (N-alkylated, amides, peptides, methionine-rich peptides and others) and to obtain information on the specificity of amino acid or short peptide side chains toward different metal ions. The complexes will be synthesized and structurally characterized by thermal and spectroscopic analysis and X-ray crystal structure diffraction experiments. The conformations of the copper complexes with amino acids will be constructed and the geometry optimized to study their possible equilibrium geometries by quantum chemical calculations.

  PROJEKTNI TIM / PROJECT TEAM

PMF, Sveučilište u Zagrebu (Faculty of Science, Univ. of Zagreb):  prof. dr. sc. Dubravka Matković-Čalogović, izv. prof. dr. sc. Draginja Mrvoš-Sermek, izv. prof. dr. sc. Biserka Prugovečki, doc. dr. sc. Ivica Đilović, doc. dr. sc. Nenad Judaš, doc. dr. sc. Ivana Biljan, dr. sc. Neven Smrečki-Lolić, mr. sc. Marina Tašner, dr. sc. Ivana Kekez, mag. chem. Darko Vušak

Institut za medicinska istraživanja i medicinu rada: dr. sc. Marijana Marković

Department of Biomedical Sciences, University of Padua: Giuseppe Zanotti,

National Institute of Chemistry, Slovenian NMR Centre: Janez Plavec

Heinrich-Heine-Universität Düsseldorf: dr. sc. Dalibor Milić

ZNANSTVENI RADOVI I SUDJELOVANJA NA KONFERENCIJAMA /

SCIENTIFIC PAPERS; CONFERENCES

https://www.bib.irb.hr/pretraga?operators=and|HRZZ-IP-2014-09-4274|text|project

USAVRŠAVANJE / PROFESSIONAL SPECIALIZATION

Darko Vušak, 15th International Symposium and Summer School on Bioanalysis, 13/07/2015 -18/07/2015, University of Medicine and Pharmacy of Targu Mures, Rumunjska

Darko Vušak, ICDD powder diffraction workshop, 23/08/2015 - 23/08/2015, Rovinj, Hrvatska

Ivana Kekez, Young Crystallographers’ Meeting 23/08/2015 -  23/08/2015, Rovinj, Hrvatska

Ivana Kekez, Dubravka Matković-Čalogović, Elettra Sincotrone Trieste, 21/09/2015 - 22/09/2015, Trst, Italija

Ivana Kekez, University of Padua, Department of Biomedical Sciences, 22/10/2015 - 23/10/2015, Padova, Italija

Ivana Kekez, Dubravka Matković-Čalogović, Ivica Đilović, Elettra Sincotrone Trieste 26/11/2015 - 27/11/2015, Trst, Italija

Ivana Kekez, University of Padua, Department of Biomedical Sciences,  20/03/2016 - 23/03/2016, Padova, Italija