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Protein synthesis in the cell is performed by large macromolecular machines called ribosomes. A general feature of the ribosomes is the large subunit stalk protuberance which consists of 4– 6 copies (2–3 dimers) of ribosomal protein L12 (P1) attached to r-protein L10 (P0). Recently, it was reported that seryl-tRNA synthetase (SerRS) interacts with P1 protein in order to recycle tRNASer molecules in archaeon Methanothermobacter thermautotrophicus [1]. Ribosomal P1 proteins feature three structural domains ; the N-terminal domain (NTD) responsible for dimer formation and binding to the ribosome, a central hinge region (linker), and a C-terminal region. The C-terminal region, composed of 18 amino acids (p18), is involved in binding of elongation factors during translation. Since the C-terminal region of P1 is highly negatively charged as tRNA, it is plausible that SerRS binds to this region. We exploited the intrinsic tryptophan SerRS fluorescence to study the interaction with P1 which does not contain tryptophans. Excitation wavelength was fixed at 295 nm, and the emission spectra was recorded from 300 to 420 nm. SerRS emission spectra show P1 concentration-dependent intensity enhancement (λmax = 335 nm) corresponding to a binding event (Kd (SerRS: P1) = (129 ± 14.2) nM). The determined affinity of the P1 deletion variant lacking 18 C-terminal amino acids (P1ΔC18) was 5-fold lower (Kd = (540 ± 79.1) nM) relative to the wild-type. This indicates that P1 NTD also participates in binding to SerRS. Interestingly, P1 mutant with linker shortened by one amino acid (P1ΔC18ΔA66) has somewhat compensatory effect on binding and the Kd was restored to (235 ± 14.6) nM. Further, we detected the binding of SerRS to the isolated free-standing C-terminal peptide (p18) confirming our hypothesis that a part of the SerRS:P1 interface involves interactions with C-terminal region as a second binding site for SerRS. These results show that P1 protein is exposing more than one binding site for SerRS because both N- and C-terminal region of P1 can contribute to the binding of SerRS. Next we established a microscale thermophoresis (MST) approach in which we studied the interaction of P1 and SerRS. In a MST- experiment a titration series of up to 16 dilutions was prepared and the highest concentration of P1 was chosen to be 20-fold higher than the expected KD. SerRS was labelled using Monolith NT™ Red Protein Labeling Kits (amine reactive) and the concentration of the fluorescently labeled SerRS was 25 nM. A serial dilution of the non-labeled titrant was prepared in the same buffer (20 mM HEPES (pH 7.5), 250 mM NaCl, 10 mM MgCl2, 5 mM β- mercaptoethanol, 0.05 % Tween-20). Mixed samples were loaded into glass capillaries and the MST-analysis was performed using the Monolith.NT115 (Nanotemper Technologies GmbH, München, Germany). Obtained dissociation constants were comparble to Kds determined by intrinsic fluorescence measurements. [1] V. Godinic-Mikulcic, J. Jaric, B. Greber, V. Franke, V. Hodnik, G. Anderluh, N. Ban, I. Weygand-Durasevic, Nucleic Acid Res 42 (2014) 5191-5201.

Protein synthesis in the cell is performed by large macromolecular machines called ribosomes. A general feature of the ribosomes is the large subunit stalk protuberance which consists of 4– 6 copies (2–3 dimers) of ribosomal protein L12 (P1) attached to r-protein L10 (P0). Recently, it was reported that seryl-tRNA synthetase (SerRS) interacts with L12 protein in order to recycle tRNASer molecules in archaeon Methanothermobacter thermautotrophicus [1]. Ribosomal L12 proteins feature three structural domains ; the N-terminal domain (NTD) responsible for dimer formation and binding to the ribosome, a central hinge region (linker), and a C-terminal region. The C-terminal region, composed of 18 amino acids, is involved in binding of elongation factors during translation. Since the C-terminal region of L12 is highly negatively charged as tRNA, it is plausible that SerRS binds to this region. We exploited the intrinsic trypthophan SerRS fluorescence to study the interaction with L12 which does not contain tryptophans. Excitation wavelength was fixed at 295 nm, and the emission spectra was recorded from 300 to 420 nm. SerRS emission spectra show L12 concentration-dependent intensity enhancement (λmax = 335 nm) corresponding to a binding event (Kd (SerRS:L12) = (129 ± 14.2) nM). The determined affinity of the L12 deletion variant lacking 18 C- terminal amino acids (L12ΔC18) was 5-fold lower (Kd = (540 ± 79.1) nM) relative to the wild-type. This indicates that L12 NTD also participates in binding to SerRS. Interestingly, L12 mutant with linker shortened by one amino acid (L12ΔC18ΔA66) has compensatory effect on binding and the Kd was restored to (235 ± 14.6) nM. It thus appears that a loose disordered full-length L12 linker destabilizes L12 NTD:SerRS interface. We hypothesize that the C-terminal region positions the linker in a specific orientation upon binding of full-lenght L12 to SerRS. In accordance, we detected the binding of SerRS to the isolated free-standing C-terminal peptide (p18) confirming our hypothesis that a part of the SerRS:L12 interface involves interactions with C-terminal region as a second binding site for SerRS. These results show that L12 protein is exposing more than one binding site for SerRS because both N- and C-terminal region of L12 can contribute to the binding of SerRS. Next we established an approach in which guanidine hydrochloride (GnHCl)-induced denaturation of a L12:SerRS complex is used to compare the stability of SerRS as a free protein or in the complex with L12. Tryptophan (Trp) fluorescence experiments report directly on the environment of the 22 Trp residues in dimeric SerRS. Upon complete unfolding, SerRS emission spectra show a red shift in emission wavelength maxima (λmax) from ∼335 nm to 355 nm. Denaturation curves over the range 0–4 M GdnHCl indicate a striking difference in stability of non-complexed and complexed SerRS. The GnHCl concentration required to obtain 50% protein denaturation (midpoint of transition) of non- complexed SerRS was 1.0 M, whereas the observed midpoint of transition for the protein in a complex was 1.9–2.0 M. Thus, L12 apparently protects SerRS from chemical denaturation. Finally, we determined the following Kd values (nM) for the L12:SerRS complex 129 ± 14.2, 205 ± 23.2, 379 ± 17.0, 126 ± 17.6 in the presence of 0.03, 0.3, 0.5 and 1 M sodium chloride, respectively. Stability of the L12:SerRS complex was not significantly perturbed in the presence of high NaCl concentrations. Methanothermobacter thermautotrophicus is a moderate halophile and requires high osmolarity for normal growth and methanogenesis. Our data agree with observation that other archaeal complexes formed by SerRS [2] can function optimally at high intracellular salt concentrations. [1] V. Godinic-Mikulcic, J. Jaric, B. Greber, V. Franke, V. Hodnik, G. Anderluh, N. Ban, I. Weygand-Durasevic, Nucleic Acid Res42 (2014) 5191-5201. [2] V. Godinic-Mikulcic, J. Jaric, C.D. Hausmann, M. Ibba, I. Weygand- Durasevic, J Biol Chem 286 (2011) 3396-3404.

Glavno područje našeg znanstvenom rada i interesa su makromolekulska udruživanja tijekom biosinteze proteina u smislu identifikacije novih proteinskih partnera, mehanizma njihovog međusobnog prepoznavanja te kvalitativne i kvantitativne analize interakcija uz pomoć fluorescencijske spektroskopije, površinske rezonancije plazmona, termoforeze i kristalografije. Budući da je kristalna struktura arhejske seril-tRNA-sintetaze (SerRS) već određena [1], pristupili smo kristalizaciji kompleksa SerRS i arginil-tRNA- sintetaze (ArgRS) jer je pokazano da upravo ova dva proteina tvore kompleks [2]. Kristalizacija ovakvog složenog sustava je vrlo zahtjevna jer su proteini velike molekule osjetljive na temperaturu, pH, ionsku jakost, prisutnost malih molekula i dr. Kako bismo dobili informacije o strukturi kompleksa, proteini su zasebno rekombinantno eksprimirani u bakteriji Escherichia coli i pročišćeni afinitetnom kromatografijom pomoću privjeska (His)6 na Zavodu za biokemiju, Prirodoslovno-matematički fakultet, Zagreb. Za potrebe rendgenske strukturne analize, proteini trebaju biti zadovoljavajuće čistoće, stoga su, prilikom pročišćavanja proteina, primijenjene metode ionsko-izmjenjivačke kromatografije, a upotrebom gel-filtracijske kromatografije dobiven je homogen uzorak koji je ukoncentriran ultrafiltracijom. Cilj istraživanja je dobiti veće kristale pravilnog oblika. Svaki proteinski sustav je jedinstven i nije moguće slijediti općenite smjernice za uspješnu kristalizaciju. Zbog toga je najprije potrebno sistematsko traženje najpovoljnijih fizikalno- kemijskih uvjeta za tvorbu kristala. Kristalizacija proteina strujanjem para je jedna od najčešće korištenih metoda za pripremu kristala proteina. Kapljica proteina se pomiješa s otopinom za kristalizaciju i stavi nad tu istu otopinu u zatvoreni bunarčić. Otopina za kristalizaciju se sastoji od precipitanta (tvar koja smanjuje topljivost proteina), soli i pufera. Korištene su metode sjedeće i viseće kapi. Proteini SerRS (dimer, Mr = 120000 ) i ArgRS (monomer, Mr = 60000) su pomiješani neposredno prije postavljanja kristalizacije u koncentraciji γ = 27 mg cm-3 te su, uz upotrebu robota za kristalizaciju The Gryphon LCP (Art Robbins Instruments), postavljene viseće kapi miješanjem 200 nl kompleksa i 200 nl kristalizacijske otopine pri 20 °C u laboratoriju profesora Nenada Bana (Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH) Zürich). Kompleksi su podvrgnuti procesu kristalizacije u ukupno 960 različitih višekomponentnih otopina za kristalizaciju. Otopine se razlikuju u vrsti i koncentraciji anorganskih soli, linearnih polimera i pH vrijednosti. Pojava prvih kristala primijećena je nakon 5 dana u kristalizacijskoj otopini koja je sadržavala 20% (w/v) polietilen-glikol (PEG, Mr = 3350) ili PEG monometil-etera (Mr = 500), kalijev izotiocijanat (c = 0, 3 - 0, 4 mol dm-3), 1, 3-bis- (tris(hidroksimetil)methilamino)-propan (c = 0, 1 mol dm-3), pH 8, 8. Određivanje trodimenzijske strukture proteina metodom difrakcije rendgenskih zraka zahtijeva pripremu jediničnog kristala proteina zadovoljavajuće veličine i kvalitete, pa nakon nalaženja početnih uvjeta kristalizacije uslijedila je optimizacija kako bi se dobili što veći i kvalitetniji kristali. Optimizacija kristalizacijskih uvjeta je uključivala variranje koncentracije proteina, precipitanta, soli, pH pufera u otopini za kristalizaciju, te temperature i veličine kapi. Pokazano je da dodatak stroncijeva klorida (c = 0, 1 mol dm-3) bitno utječe na kvalitetu kristala. [1] S. Bilokapic, T. Maier, D. Ahel, I. Gruic- Sovulj, D. Söll, I. Weygand-Durasevic, N. Ban. EMBO Journal (2006) 25:2498-2509. [2] V. Godinic-Mikulcic, J. Jaric, C.D.Hausmann, M. Ibba, I. Weygand-Durasevic. Journal of Biological Chemistry (2011) 286:3396-3404.

Pripravljeni su novi derivati kaliks[4]arena s različitim dietilenglikolskim lancima na donjem obodu kaliksarenskog prstena te je istraženo njihovo vezanje s kationima alkalijskih i zemnoalkalijskih metala. Sintetizirana su dva sekundarna amidna derivata (5, 11, 17, 23- tetra-tert-butil-25, 26, 27, 28-tetra(N-(2-(2- hidroksietoksi)etil)karbamoilmetoksi)kaliks[4]a ren (1) i 5, 11, 17, 23-tetra-tert-butil-25, 26, 27, 28-tetra(N-(2-(2- metoksietoksi)etil)karbamoilmetoksi)kaliks[4]ar en (2)) te jedan tercijarni amidni derivat (5, 11, 17, 23-tetra-tert-butil-25, 26, 27, 28- tetra(N, N-bis(2-(2-metoksietoksi)etil) karbamoilmetoksi)kaliks[4]aren (3)). Spojevi su okarakterizirani na temelju podataka dobivenih 1H i 13C NMR te FTIR spektroskopijom, kao i spektrometrijom masa. Kompleksacijski afiniteti liganada 1–3 prema kationima alkalijskih i zemnoalkalijskih metala u acetonitrilu i metanolu istraženi su primjenom spektrometrijskih (UV, NMR) i elektrokemijskih (potenciometrija, konduktometrija) metoda. Obradom titracijskih podataka izračunane su (u nekim slučajevima samo procijenjene) konstante stabilnosti odgovarajućih kompleksa stehiometrije 1:1 (kation:ligand) pri 25 °C. Vrijednosti konstanti stabilnosti određene raznim metodama dobro se slažu. Uočena je mnogo veća stabilnost kompleksa s tercijarnim amidnim derivatom (3) u odnosu na one sa sekundarnim amidnim derivatima (1, 2), što upućuje na utjecaj intramolekulskih vodikovih veza na kompleksacijska svojstva pripravljenih derivata kaliks[4]arena. Opaženo je da otapalo znatno utječe na stabilnost kompleksa te da važnu ulogu u reakciji kompleksiranja kationa igra njegova solvatacija. Svi istraženi kaliksareni pokazuju veći afinitet prema kationima u acetonitrilu nego u metanolu. U većini slučajeva ligandi bolje vežu katione zemnoalkalijskih u odnosu na alkalijske metale. Istražena je i ekstrakcija metalnih pikrata iz vode u diklormetan putem kompleksiranja metalnih iona s ligandima 1–3. Svi kationi prilično se dobro ekstrahiraju pomoću tercijarnog amidnog derivata dok se pomoću sekundarnih amidnih derivata ekstrahiraju umjereno samo natrijev, kalijev i kalcijev kation.

Our previous work was focused on systematic research of synthesis and analysis of heterocyclic molecules. As a part of that research, present work continues the characterization of various bioactive derivatives of pyridines and their metal complexes. Pyridine derivatives have interesting pharmacological properties and are reported to possess many biological activities such as antibacterial, antifungal, antiviral and antitumor. The type of biological activity is influenced by pyridine ring substituents. 2- hydrazino-6-methyl-4-(methoxymethyl)-5- nitropyridine-3-carbonitrile was synthesized and confirmed by single crystal X-ray diffraction method and corresponding molecular structure was experimentally characterized by means of IR, 1H- NMR, 13C-NMR and UV-vis spectroscopy techniques. UV-vis absorption and emission spectra of the compound were recorded in protic and aprotic solvents at 25 °C. Spectra of aqueous solution were also recorded at different temperatures and pH values and protonation constant was calculated.

Priređeni su i ispitivani kompleksi vanadija(IV) sa salicilaldehid tiosemikarbazonom i 4– fenilsalicilaldehid tiosemikarbazonom, pri različitim reakcijskim uvjetima (sobna i povišena temperatura, reakcije in situ, dodatak jakih elektron donora). Izolirani su sljedeći kompleksni spojevi: 1. oksobis(salicilaldehidtiosemikarbazonato)vanadij(IV), [VO(TSCSal)2] · 3/2CH3OH, 2. oksobis(salicilaldehidtiosemikarbazonato)vanadij(IV), [VO(TSCSal)2] · 3/4(γ – pikolin), 3. metanolokso– 4– fenilsalicilaldehidtiosemikarbazonatovanadij(IV), [VO(4– PhTSCSal)(CH3OH)]. Dobiveni kompleksni spojevi identificirani su na osnovi podataka dobivenih instrumentnim metodama: infracrvena spektroskopija, termogravimetrijska analiza i roentgenska strukturna analiza te kemijske analiza ugljika, vodika, dušika i sumpora. Sastav trećeg kompleksnog spoja potvrđen je i roentgenskom strukturnom analizom na monokristalnom uzorku.

Affinity chromatography is a powerful purification method based on specific interaction of an e.g. enzyme and substrate, receptor and ligand, or antibody and antigen (immunoaffinity chromatography). Advantages of immunoaffinity chromatography are that it can be used for any protein (or even virus) regardless of its function and a very high binding affinity of antigen and antibody enabling isolation of a highly pure interacting partner. Disadvantage of the immunoaffinity chromatography is that a specific competitive elution cannot be employed as is the case with affinity chromatography employing e.g. enzyme or a receptor. Also, high affinity antigen- antibody interactions can only be disrupted using extreme conditions such as low pH conditions, denaturating agents or chaotropic salts. These conditions affect all noncovalent interactions, not only intermolecular but also intramolecular, potentially causing damage to the immobilized protein and the protein to be isolated making immunoaffinity inappropriate for general use. We aimed to find elution conditions disrupting antigen-antibody interactions but under native conditions. For testing of these elution conditions we used two immunoaffinity columns. In both cases columns were prepared by binding of antibodies to epoxy-activated monolith column. One column was specific for ovalbumin and the other for mumps virus. For comparison we also used tested elution systems with commercial protein G column and immunoglobulin solution. Effectiveness of tested elution systems was found different in these three columns, but the trend was the same. This indicates that possibly the differences in dissociation constants i.e. interaction strength are reflected in the obtained results regarding effectiveness of tested elution systems. So we would like to determine dissociation constant of ovalbumin-anti-OVA-antibody and to compare it with dissociation constant of protein G- immunoglobulin couple. Also, we would like to determine dissociation constants of mumps and measles viruses with their respective antibodies to gain insight into these interactions also.

Aminoacyl-tRNA synthetases (aaRS) attach appropriate amino acids to cognate tRNAs ensuring efficient protein biosynthesis. Their involvement in diverse celullar functions beyond translation opens broad new perspectives in functional proteomics, especially in the field of plant aaRSs which is fairly uninvestigated. To shed light on non-canonical functions of cytosolic seryl-tRNA synthetase (SerRS) from plant Arabidopsis thaliana we conducted yeast two hybrid screen (Y2H) which revealed metabolic protein BEN1 as a highly promising interacting partner. Interaction was further biophysicaly analyzed in vitro using isothermal calorimetry titration (ITC), pull- down, surface plasmon resonance (SPR) and microscale thermophoresis method (MST). Due to the nature of interaction and sensitivity of applied methods we were not able to retrieve positive results using pull-down assay and ITC, but SPR and MST gave us postive confirmation of interaction and information about dissociation constant (Kd constant obtained using SPR = 1, 03 × 10-6 (± 1, 67 × 10-7) mol dm-3, in good agreement with Kd obtained using MST = 4, 45 × 10-7 (± 1, 40 × 10-7) mol dm-3). To determine interaction surfaces and pinpoint regions responsible for SerRS and BEN1 interaction, truncated variants of both SerRS and BEN1 proteins were prepared and protein interactions were analyzed using MST. Kd for complex containing BEN1 and truncated SerRS variant without basic C-terminal extension (with or without (His)6 tag) was similar to Kd of the SerRS:BEN1 complex indicating that SerRS basic C- terminal extension does not have any influence on interaction. Furthermore, isolated N-terminal domain of SerRS did not form the complex with BEN1. Taking into account observed data, we concluded that interaction between SerRS and BEN1 involves the central part of SerRS that contains globular catalytic domain. Determining interaction domain of BEN1 was not possible because of aggregation problems in MST assay when using truncated BEN1 version, so this part of investigation is yet to be done. Knowing precise interaction contacts will allow us to understand and propose functional importance of SerRS:BEN1 assembly in plant organism.

Aminoacyl-tRNA synthetases (aaRSs) covalently pair cognate amino acid and cognate tRNA in an ATP- dependent manner. Due to structural similarity among different amino acids, some aaRSs also catalyze synthetic reactions with noncognate amino acids. To maintain accuracy of translation, these aaRSs have evolved proofreading mechanisms. The more widespread mechanism is hydrolysis of noncognate aminoacyl-adenylate called pre-transfer editing, occurring in the synthetic site. More efficient mechanism is post-transfer editing or deacylation of mischarged tRNA, which is occurring in a separate domain. To prevent futile ATP consumption, the deacylation domain efficiently discriminates against cognate charged tRNA. Here we explored interaction of leucyl-tRNA synthetase (LeuRS) with uncharged tRNALeu as its substrate and further with cognate and noncognate charged tRNA (Leu- tRNALeu and NvatRNALeu), which are products of synthetic reactions, but substrates for the hydrolytic reaction by LeuRS. Microscale themophoresis was valuable tool for studying these interactions due to its speed and requirement for small amount of material. Combining these results with the other thermodynamic and kinetic approaches demonstrated that substrate specificity in deacylation is predominately governed by the complementarity established at the transition state.

Simpozij i radionica o termoforezi održana je 30.6. i 1.7.2015. na Kemijskom odsjeku PMF-a Sveučilišta u Zagrebu u okviru projekta FP7 IntegraLife. Skup je sadržavao predavanja, praktičnu radionicu i postersku sekciju. Na skupu je sudjelovalo 67 sudionika, od toga 42 u praktičnoj radionici. Održano je 6 predavanja i prikazano 17 posterskih priopćenja.

It has been estimated that over 80% of proteins do not operate alone but in complexes. Examples of important complexes are the spliceosome, the ribosome and the nuclear pore complex. The ribosome is a ribonucleoprotein complex that translates the genetic information in the cell into the encoded polypeptides. The fidelity with which mRNA is decoded into proteins is essential for maintenance of the genetic code and it is highly dependent on the specific attachment of amino acids to tRNAs by aminoacyl-tRNAsynthetases (aaRSs). Multi- aminoacyl-tRNAsynthetase complexes of different compositions occur throughout the tree of life. To further investigate the extent and composition of multi-aaRS complexes in archaea we have undertaken a yeast two-hybrid search for proteins that interact with methanogenic- type SerRS (mSerRS), an atypical form of SerRS confined to certain archaea such as Methanothermobacter thermautotrophicus (M. thermautotrophicus). Yeast two-hybrid screens revealed a interaction between two aaRSs: mSerRS and ArgRS, which appears to specifically enhance tRNASer aminoacylation. Moreover, in the same organism, few ribosomal proteins were found also associated with mSerRS. Chemical crosslinking and surface plasmon resonance (SPR) experiments confirmed that mSerRS and ArgRS associate with large ribosomal subunit proteins (1). Microscale thermophoresis (MST) was used to determine dissociation constant (Kd) for the fluorescently labeled ribosomal particles in interaction with mSerRS and MtArgRS consistent with formation of a stable macromolecular complex detected by ultracentrifugation. A general feature of the ribosome is the large subunit stalk protuberance which consists of 4–6 copies (2–3 dimers) of ribosomal protein P1 (L12) where the elongation factors bind and act. We consider ribosomal protein P1 to be a primary target for interaction of mSerRS with the ribosome and thus we exploited the intrinsic trypthophan SerRS fluorescence to study the interaction with wild type and mutant variants of L12 by fluorescence spectroscopy. Obtained dissociation constants for mSerRS:L12 complexes were comparable to Kds determined by MST analysis. In addition, the assembly of mSerRS with M. thermautotrophicus ribosome is supported by analysis of the ordering of synonymous codons. In M. thermautotrophicus found that the codon choice at the next instance of the same amino acid may be influenced by the previous codon upstream, causing autocorrelation of codon pairs read by the same tRNA. Therefore, the association of components of the protein synthesis machinery into macromolecular assemblies has the potential to increase translational efficiency by limiting substrate diffusion away from the ribosome thereby allowing rapid recycling of tRNAs. [1] Godinic-Mikulcic, Vlatka ; Jaric, Jelena ; Greber, Basil ; Franke, Vedran ; Hodnik, Vesna ; Anderluh, Gregor ; Ban, Nenad and Weygand- Durasevic, Ivana (2014) Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs. Nucl. Acids Res. 42 (8):5191- 5201.