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Title: Mutagenesis studies on the membrane anchoring properties of human CD2
Author: Corcao, Gertrudes
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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There is a lack of information about structural requirements for a functional transmembrane domain in eukaryotic membrane proteins. This led me to design a eukaryotic system to bring more information about this structure and the role of the positively charged residues situated at the cytoplasmic side of a transmembrane region. The CD2 molecule was chosen as a model for an integral membrane protein. I deleted the transmembrane domain (26 amino acids) by oligonucleotide site-directed mutagenesis and overlap extension using PCR mutagenesis. Truncated forms with transmembrane regions 14, 12, 10 and 8 amino acids long were created. The common positive cluster (Lys-Arg-Lys-Lys) at the cytoplasmic domain was disrupted by substituting it for polar residues (Gln-Gln-Gln-Gln). The effects of such mutagenesis was examined after expression of the mutant proteins in eukaryotic cells (COS-7 and CHO) . Cellular localization was determined by panning and immunostaining experiments. The functional state of the mutant proteins expressed on the cell surface was verified by resetting experiments and immunostaining with antibodies against different epitopes of the CD2 extracellular domain. The interaction of the molecule with the lipid bilayer was determined by lateral diffusion studies, using photobleaching analysis. It was demonstrated that a transmembrane domain at least 12 amino acids long is necessary for the CD2 protein to be expressed on the cell surface as an integral membrane protein. Proteins with shorter transmembrane domains (10 and 8 amino acids respectively) were localized intracellularly as verified by immunostaining. This finding is in agreement with previous work on the VSV G protein (Adams and Rose, 1985b) . Lateral diffusion studies revealed that even large deletions in the transmembrane domain of CD2 do not interfere with its lateral mobility in the lipid bilayer. Although the fraction of the free molecules for diffusion was higher in the CD2 protein with transmembrane region 12 amino acids long. This implies that deletions in the transmembrane domain can interfere with the stability of the protein within the lipid bilayer. The disruption of the positively charged cluster of the CD2 cytoplasmic domain did not alter the orientation and stability of the molecule within the cell membrane. This suggests that the topology of a membrane protein with only one span domain is not influenced by this positively charged sequence. The orientation within the membrane should be determined in the early stages of translocation by interaction of the signal sequence and the hydrophobic domain with the protein-conducting pore in the endoplasmic reticulum membrane.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available