A biochemical analysis into the co-translational folding of a G protein-coupled receptor; GPR35

The folding and targeting of membrane proteins poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane (TM) domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer. The biogenesis of a mature membrane protein takes place through the insertion and integration into the lipid bilayer. A number of TM proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. Although studies into the folding and targeting of a number of membrane proteins have been carried out to date, there is little information on one of the largest class of eukaryotic membrane proteins; the G-protein-coupled receptors (GPCRs). This project studies the early folding events of the human ortholog of GPR35. To analyse the structure of the 1st TM domain, intermediates were generated and assessed by the biochemical method of pegylation (PEG-MAL). A structurally-similar microbial opsin (Bacterioopsin) was also used to investigate the differences in the early protein folding within eukaryotic and prokaryotic translation systems. Results showed that neither the 1st TM domain of GPR35 nor Bacterioopsin were capable of compacting in the ribosome tunnel before their N-terminus reached the ribosome exit point. The results for this assay remained consistent whether the proteins were translated in a eukaryotic or prokaryotic translation system. To examine the communication mechanism between the ribosome, the nascent chain and the protein targeting pathway, crosslinking experiments were carried out using the homobifunctional lysine cross-linker BS3. Specifically, the data generated here show that the nascent chain of GPR35 reaches the ribosomal protein uL23 in an extended conformation and interacts with the SRP protein as it exits the ribosome tunnel. This confirms the role of SRP in the co-translational targeting of GPR35. Using these methods insights into the early folding of GPCRs has been obtained. Further experiments using site-directed mutagenesis to reduce hydrophobicity in the 1st TM domain of GPR35, highlighted the mechanisms by which GPCRs are targeted to the endoplasmic reticulum. Confirming that hydrophobicity within the signal anchor sequence is essential of SRP-dependent targeting. Following the successful interaction of the nascent GPR35 and SRP, GPR35 is successfully targeted to ER membranes, shown here as dog pancreas microsomes (DPMs). Glycosylation of the GPR35 N-terminus was used to determine nascent chain structure as it is inserted into the ER membrane. These glycosylation experiments confirm that TM1 has obtained its compacted state whilst residing in the translocon. Finally, a site-specific cross-linking approach using the homobifunctional cysteine cross-linker, BMH, was used to study the lateral integration of GPR35 into the ER. Cross-linking of GPR35 TM1 and TM2 could be detected adjacent to a protein of ~45kDa, believed to be Sec61α. The loss of this adduct, as the nascent chain extends, showed the lateral movement of GPR35 TM1 from the translocon was dependent on the subsequent synthesis of TM2.

Synthetic biology in droplet-based microfluidics

Droplet microfluidics is an active multidisciplinary area of research that evolved out of the larger field of microfluidics. It enables the user to handle, process and manipulate micrometer-sized emulsion droplets on a micro- fabricated platform. The capability to carry out a large number of individual experiments per unit time makes the droplet microfluidic technology an ideal high-throughput platform for analysis of biological and biochemical samples. The objective of this thesis was to use such a technology for designing systems with novel implications in the newly emerging field of synthetic biology. Chapter 4, the first results chapter, introduces a novel method of droplet coalescence using a flow-focusing capillary device. In Chapter 5, the development of a microfluidic platform for the fabrication of a cell-free micro-environment for site-specific gene manipulation and protein expression is described. Furthermore, a novel fluorescent reporter system which functions both in vivo and in vitro is introduced in this chapter. Chapter 6 covers the microfluidic fabrication of polymeric vesicles from poly(2-methyloxazoline-b-dimethylsiloxane-b-2-methyloxazoline) tri-block copolymer. The polymersome made from this polymer was used in the next Chapter for the study of a chimeric membrane protein called mRFP1-EstA∗. In Chapter 7, the application of microfluidics for the fabrication of synthetic biological membranes to recreate artificial cell- like chassis structures for reconstitution of a membrane-anchored protein is described.