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Assembling biomembranes: fundamentals of membrane transporter folding and creation of synthetic modules

Final Report Summary - MEMSEMBLE (Assembling biomembranes: fundamentals of membrane transporter folding and creation of synthetic modules)

Membranes surround cells and subdivide the cells into compartments, with membrane-embedded proteins allowing information and matter to pass across the membranes and between cells. The human genome sequence carries the code for the proteins in my bodies. We have yet to identify what each gene means, which protein it encodes and how the genetic information is translated into a functional protein. The most glaring gaps in my knowledge come with membrane proteins, which constitute about a third of the proteins in my bodies, and well over half of the current targets in the development of new medicines. This grant aimed to find out how genetic information is translated to make membrane proteins. Genes are first decoded to give a string of amino acids, which then has to “fold” to the protein’s correct, and unique, three-dimensional shape. Such folding beautifully illustrates natural self-assembly. This work progressed understanding of biological construction and mimic it in the bioengineering of tuneable synthetic systems. I have devised methods to study the folding of several types of membrane protein and have used the knowledge gained to begin to construct synthetic modules that can manufacture the proteins. These will allow bespoke miniature devices to be assembled that form the basis of artificial cells.
The progress made on this grant relied on the development of new experimental methods. No one method can provide all the answers to the complex biosynthetic process of making a membrane protein in a cell. I have exploited and adapted some promising powerful analytical techniques that involve Infra Red spectroscopy, which provides a fingerprint of protein structure formation during folding, as well as a mass spectrometry approach that allows the motions of the protein to be determined. I also developed a range of experimental samples that have allowed me to probe the folding of the protein in defined, controllable conditions as well as those that mimic the cellular environment.

The main conclusions from the study relating to membrane protein folding are that membrane mechanical properties modulate folding, both in synthetic systems as during co-translational folding as the protein is being synthesised on the ribosome. Membranes are two-dimensional liquid crystals with particular elastic, mechanical properties. I have shown that the intrinsic stability of a folded protein is coupled to membrane mechanical properties. Moreover, I have demonstrated that membrane mechanics can also modulate co-translational folding to the extent that efficient folding can occur without the assistance of the cellular translocon apparatus that is used to aid insertion in vivo. The membrane control mechanism may also reveal the basis of an important regulatory mechanism for membrane protein biosynthesis - in that different membranes have contrasting properties that will influence the various stages of insertion, folding and function in different ways. Thus cells may manipulate their local composition at different stages of folding to ensure correct folding and avoid the damaging consequences of misfolding.

There are also significant conclusions relating to artificial cell and synthetic tissue aspects of synthetic biology. The main advances are in relation to de novo cell construction. Artificial membranes at the junction of two cellular droplets have emerged as a method of choice for constructing artificial cells and tissues. But, the artificial membrane needs to be functionalised; thus far only passive, diffusion controlled, ungated transport of molecules has been demonstrated between the droplets. These methods cannot sustain or control reactions for cellular mimics. I have demonstrated the first specific transport of a metabolite across the artificial membrane and shown this can be used to build up the concentration of the metabolite in the artificial cell droplet. I have additionally synthesised the relevant transport protein in situ, from its gene in the droplet, thus furthering the goal of a self synthesising, self assembling cellular mimic.