DNA & Protein Wet Lab
MSROP2016: Effects of Interaction Between Tyrosine and Methionine on Methionine Oxidation and Fibrillation of Alpha Synuclein
Proposal:
Alpha-synuclein is a pre-synaptic protein, which is proposed to be directly related to Parkinson’s Disease and dementia with Lewy bodies. Pathological morphology of this protein usually consists of beta-rich sheet amyloid fibrils, which further contributes to the formation of Lewy bodies. It has been proposed previously that oxidation of Methionines (especially Met127) has direct effects on alpha-synuclein’s aggregation, and that interaction between Methionines and nearby Tyrosines may also influence this oxidation process.
In order to initiate an examination of the mentioned proposals, this project aims at mutating within the protein’s amino acid sequence, the following Tyrosine residues including Tyr125, Tyr39, Tyr133, and Tyr136 into Alanine residues using site-directed mutagenesis techniques. At the end of the project, bacterial glycerol stocks of mutant plasmid with confirmed sequence are produced, stored appropriately, and ready for future protein purification process and fibrillation detection assay (ThioflavinT, fluorescence, etc.).
Report:
Directed Research Course: Roles of Disulfide-linked Dimer in Transmembrane Domain of TNFR1
Proposal:
Tumor necrosis factor receptor 1 (TNFR1) is a member of the TNFR superfamily. It comprises an N-terminal extracellular domain (ECD), a transmembrane domain, and a cytoplasmic death domain that is crucial for initiation of death signaling upon binding of one of two ligands, tumor necrosis factor (TNF-a or lymphotoxin-alpha (LT-a). The extracellular domain consists of four cysteine-rich domains (CRD). Crystal structures have shown that TNFR1 exists as dimer. This dimerization is largely attributed to the interaction at the pre-ligand assembly domain (PLAD), which is distal from the membrane of the receptor. Interestingly, the transmembrane domain of TNFR1 also has a cysteine at position 223, which may be capable of forming disulfide bond with TM-domain of neighboring TNFR1. However, the existence of disulfide-linked dimer in TM-domain of TNFR1 is not explored.
In this research project, my goal is to answer the following questions: 1) Is there any disulfide -linked dimer in the transmembrane domain of TNFR1? ; 2) If yes, is the formation of this dimer is ligand-dependent? 3) Is this transmembrane dimer play role in the downstream signaling of TNFR1? These goals can be achieved by using site-directed genesis, western blot, and cell viability assay.
Report:
WET LAB PROTOCOLS
- DNA Cloning (restriction, extraction, ligation, transformation)
- Protein Purification
- Western blot protocol (sample prep, SDS- Page, membrane transfer, immunoblotting, detection)