Naslov
Analysis of protein interactions using fluorescence technologies

Autori
Godinić Mikulčić, Vlatka ; Sviben, Igor ; Janeš, Karla ; Kveštak, Martina ; Rokov-Plavec, Jasmina ; Gruić-Sovulj, Ita

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts of the Symposium and Workshop on Microscale Thermophoresis / Godinić Mikulčić, Vlatka ; Sviben, Igor ; Rokov-Plavec, Jasmina - Zagreb : Prirodoslovno-matematički fakultet Sveučilišta u Zagrebu, 2015, 18-18

ISBN
978-953-6076-37-6

Skup
Symposium and Workshop on Microscale Thermophoresis

Mjesto i datum
Zagreb, Hrvatska, 30.06.2015. - 01.07.2015

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
protein interactions; fluorescence spectrophotometry; microscale thermophoresis; dissociation constant; protein synthesis; ribosome

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

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