PhD Scientific Days

Poster Presentation: Basic Sciences

P63: Production and NMR examination of the interface domain-deleted human dihydrolipoamide dehydrogenase

Előadó neve

Nagy, Balint

Előadó munkahelye

Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest

Előadó telefonszáma

36706305520

Előadó e-mail címe

nagy.balint@med.semmelweis-univ.hu

Az előadás címe

Production and NMR examination of the interface domain-deleted human dihydrolipoamide dehydrogenase

Szerző(k) neve és munkahelye

Balint Nagy, Agnes Hubert, Zsofia Zambo, Vera Adam-Vizi, Attila Ambrus
Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest

Szekció

Poster Presentation: Basic Sciences

Data of the presenter

Doctoral School: János Szentágothai Ph.D. Doctoral School of Neurosciences
Program: Functional Neurosciences Program
Supervisor: Attila Ambrus
E-mail address: nagy.balint@med.semmelweis-univ.hu

Text of the abstract

INTRODUCTION: The human α-ketoglutarate dehydrogenase complex (hKGDHc) has a key role in the energy production of cells by catalyzing an irreversible step in the Krebs cycle. Under certain pathological conditions hKGDHc becomes a significant source of reactive oxygen species (ROS) and is proposed to have a fundamental role in the pathogeneses of several neurological and cardiological disorders. ROS are generated by the intact hKGDHc at the E3 component (dihydrolipoamide dehydrogenase, hLADH).
AIM: The main focus of our research is to understand the molecular mechanism of ROS generation by hLADH. In this study, structure of a catalytically important segment of hLADH is investigated using Nuclear Magnetic Resonance (NMR) spectroscopy.
METHODS: The gene segment encoding the dimerization interface domain-deleted (DIDD) hLADH was codon-optimized for E. coli expression, cloned into the pET52b+ vector, and expressed in E. coli BL21(DE3). The protein was produced with a double Strep affinity tag on the N-terminus and was purified to homogeneity in a single affinity chromatography step after successful refolding from inclusion bodies. 15N-labeled protein was expressed in minimal medium containing 15N-NH4Cl.
RESULTS: DIDD-hLADH (36 kDa) was successfully expressed, refolded from inclusion bodies under optimized conditions, and purified. The sequence and proper folding of the protein were verified by mass spectrometry and circular dichroism spectroscopy, respectively. 15N-DIDD-hLADH displayed a promising signal dispersion, but an overall spectral behavior that is not yet adequate for structure determination, in 1H-15N correlation NMR experiments at 800 MHz of proton.
CONCLUSIONS: The functional homodimer of hLADH (100 kDa) is unavailable for NMR techniques. Deletion of the dimerization interface domain might help overcome this hurdle. Cleavage of the affinity tag might also improve the spectral behavior. After successful optimizations with 15N-DIDD-hLADH, 13C/15N(/2H)-labeled protein will be produced for structure determination.

Azonosító

P63

Kind

Szabad

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

1331

Authors (legacy)