PhD Scientific Days

Poster Presentation: Neurosciences

P55: Structural investigation of the human alpha-ketoglutarate dehydrogenase complex

Előadó neve

Zámbó, Zsófia

Előadó munkahelye

Semmelweis University, Department of Medical Biochemistry

Előadó telefonszáma

+36-20-6660049

Előadó e-mail címe

zambo.zsofia@med.semmelweis-univ.hu

Az előadás címe

Structural investigation of the human alpha-ketoglutarate dehydrogenase complex

Szerző(k) neve és munkahelye

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

Szekció

Poster Presentation: Neurosciences

Data of the presenter

Doctoral School: János Szentágothai Doctoral School of Neurosciences
Program: Functional Neurosciences
Supervisor: Dr. Attila Ambrus
E-mail addres: zambo.zsofia@med.semmelweis-univ.hu

Text of the abstract

Introduction: The alpha-ketoglutarate dehydrogenase complex (KGDHc) is a rate-limiting enzyme in the Krebs cycle and it consists of multiple copies of three subunits (E1-3). The hKGDHc can produce significant amounts of reactive oxygen species (ROS) under certain pathological conditions. The dysfunction and ROS generation of the hKGDHc are heavily implicated in oxidative stress and related pathologies (e.g. neurodegenerative disorders).
Aims: We intended to isolate the recombinant components of the hKGDHc and generate various mutants of hE3. All these protein products will be used for biochemical and structural analyses.
Methods: Genes were codon-optimized for E. coli expression, cloned into the pET52b+ plasmid, and expressed in E. coli BL21(DE3). Side-directed mutagenesis was carried out using the QuikChange II kit. The constructs included single or double Strep affinity tags on either or both protein termini for purification in a single step. Structural elucidation involved circular dichroism (CD) spectroscopy, mass spectrometry, x-ray crystallography, and cryo-electron microscopy (cryo-EM).
Results: 14 pathogenic, 20 active site, and 12 His-to-Ala hE3 mutants and the hE2 were successfully purified to homogeneity. Sequence identity of the protein products was verified by mass spectrometry. CD signatures of selected hE3 mutants indicated altered conformations relative to hE3. The hE2 component exhibited a higher order oligomerization state (likely a 24-meric structure) according to cryo-EM. A hE3 pathogenic mutant was successfully crystallized and its synchrotron x-ray analysis was performed.
Conclusion: The homomultimeric form of the hE2 component appears to be an ideal candidate for further cryo-EM analysis. Cryo-EM studies of the reconstituted hKGDHc may also be attainable in the future. Mass spectrometry revealed mixtures of hE3 mutants in selected preparations, which warrants attention, further development of the purification protocol, and new expression of the respective mutants. Due to the predominant formation of inclusion bodies the purification of hE1 requires further optimization.

Azonosító

P55

Kind

Szabad

Előadás fájl jóváhagyás

nem rendelkezett róla

Előadó

1253

Authors (legacy)